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Effect of age on the binding of lectin 125I-PHA-B to pancreatic islets of rat in vitro and stimulation of some cellular events.

Agaricus bisporus lectin (PHA-B) stimulates insulin release in vitro. The stimulation is associated with increased conversion of proinsulin to insulin in the isolated islets of Langerhans of rats. Both these functions are directly proportional to the binding of I125 PHA-B, which is more marked in the islets from younger rats. The lectin binding to islets is not affected by glucose concentration in the medium.

Agaricus↗

Effect of age on Agaricus bisporus PHA-B stimulated insulin release and 45Ca2+ uptake in vitro by islets of Langerhans.

The in vitro basal insulin release and 45Ca2+ uptake by rat islets of Langerhans remain unaffected by the animal's age. However, when Agaricus bisporus lectin PHA-B is added to the medium, there is a significant stimulation of basal insulin secretion and 45Ca2+ uptake. The islets isolated from younger rats are more sensitive to A. bisporus PHA-B stimulation and register 1.5-fold and 3-fold more increases of insulin release and 45Ca2+ uptake respectively as compared to islets isolated from 12-month-old rats. Age-related conformational changes in the B-cell membrane are suggested as a possible explanation for the above observation.

Aging↗

Effect of PHA-B fraction of Agaricus bisporus lectin on insulin release and 45Ca2+ uptake by islets of Langerhans in vitro.

The effect of a purified PHA-B fraction of Agaricus bisporus lectin on insulin release and 45Ca2+ uptake by islets of Langerhans was studied. It was found that at 2 mM glucose +17.5 micrograms/ml A. bisporus PHA-B (which produces half-maximal stimulation) there was a 3.3 fold increase in insulin secretion as compared to controls (2 mM glucose). Stimulation of insulin release was found to be dose-related as well as time-dependent. Maximal stimulation of insulin release was obtained at a lectin concentration of about 60 micrograms/ml and proceeded as a linear function of time for about 1 h. It was found that in the presence of 2 mM glucose +17.5 micrograms/ml lectin islet 45Ca2+ uptake was enhanced more than 11-fold as compared to controls. 45Ca2+ uptake was found to be lectin dose- and time-dependent and maximal uptake was achieved at a lectin concentration of about 60 micrograms/ml after which the rate slowed down until it reached a stationary phase at about 75 micrograms/ml.

Animals↗

Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs.

Medium-chain-length poly-3-(R)-hydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The minimum gene-set for the accumulation of mcl-PHAs from de novo fatty acid biosynthesis has been identified in prokaryotes as consisting of the Pha-C1 polymerase and the ACP-CoA-transacylase. In this paper, the synthesis of mcl-PHAs has been attempted in transgenic potato (Solanum tuberosum L.) using the same set of genes that were introduced into potato by particle bombardment. Polymer contents of transgenic lines were analysed by gas chromatography and by a new simple method employing a size-exclusion filter column. The expression of the Pha-C1 polymerase and the ACP-CoA-transacylase in the plastids of transgenic potato led to the synthesis of a hydrophobic polymer composed of mcl-hydroxy-fatty acids with carbon chain lengths ranging from C-6 to C-12 in leaves of the selected transgenic lines. We strongly suggest that the polymer observed consists of mcl-PHAs and that this report establishes for the first time a possible route for the production of mcl-PHAs from de novo fatty acid biosynthesis in plants.

Acyltransferases↗

Alpha-fetoprotein gene expression in human lymphoblastoid cells and in PHA-stimulated normal T-lymphocytes.

Alpha-fetoprotein (AFP) is mainly synthesized by the fetal liver, the yolk sac and, to a much lower extent, by a few non-hepatic fetal tissues (i.e. kidney, pancreas, lung). This property is considered to be lost in mature quiescent cells of the adult. In the present we have studied the expression of AFP mRNA sequences in phytohemagglutinin (PHA)-stimulated normal human T-lymphocytes and in several human lymphoma cell lines. The amount of mRNA transcripts detected in quiescent T-lymphocytes by dot and Northern blot analysis was very low. It increased rapidly after PHA-activation, reached a maximum at 72 hours (six fold the level observed for quiescent T-lymphocytes) and decreased thereafter. The lymphoma cell lines Daudi, Raji, Rh6 et CEM, all expressed elevated levels of AFP mRNA. The transcripts had the size expected for human AFP, suggesting that they were functional and probably translated into protein. The possible role of AFP synthesis in lymphocyte blastogenesis and in lymphoma growth is discussed in relation with the strong binding affinity of this protein for polyunsaturated fatty acids.

DNA Probes↗

Light microscopic evidence of striatal input to intrapallidal neurons of cholinergic cell group Ch4 in the rat: a study employing the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L).

Injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) were placed in various striatal loci in the rat. Within the globus pallidus, PHA-L-filled striatofugal axons were seen to approach cholinergic neurons, identified with either acetylcholinesterase histochemistry or choline acetyltransferase immunohistochemistry, and, apparently, to contact the surface of such cells with axonal varicosities. Since these varicosities are thought to mark the sites of synaptic terminals, such juxtapositions provide strong light-microscopic evidence that intrapallidal cholinergic neurons in the rat receive a direct innervation from the striatum and are integrated into the circuitry of the basal ganglia.

Acetylcholinesterase↗

PHA-L can be transported anterogradely through fibers of passage.

Iontophoretic injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) were restricted to the dorsal columns in the spinal cords of 9 rats. In 5 of these, labeling was found in the dorsal column nuclei, indicating that fibers of passage can transport PHA-L. However, labeled fibers were less dense and less intensely stained than those seen following similar injections that involved the dorsal horn.

Animals↗

Climbing fibers are labelled after injection of PHA-L into the nucleus interpositus of the cat.

After injection of Phaseolus vulgaris-leucoagglutinin (PHA-L) into the interposed nucleus of the cat, labelled fibers were found in the granular layer as well as in the molecular layer of the cerebellar cortex. Those in the molecular layer were judged from their location and morphology as climbing fibers. Such labelling of climbing fibers which have not been labelled by autoradiography could be attributed to higher sensitivity and resolution of PHA-L method. In addition, Purkinje cell soma and its dendrites were labelled in a narrow stripe in the vermal and paravermal cortex. The pattern of labelling suggests that they were labelled by retrograde transport of the lectin.

Animals↗

Ascending and descending projections of the nucleus reticularis gigantocellularis in the cat demonstrated by the anterograde neural tracer, Phaseolus vulgaris leucoagglutinin (PHA-L).

Ascending and descending projections of the nucleus reticularis gigantocellularis (NRGc) were studied in the cat by the anterograde tracer, Phaseolus vulgaris leucoagglutinin. Ascending fibers from the left or the right NRGc coursed through the bilateral medial reticular formation and some of them reached the diencephalon. In the brainstem, PHA-L-labeled fibers and their terminals were observed in the medial reticular formation, the cranial motor nuclei (III, IV, V, VI, VII, XII), the vestibular complex, the LC complex, the raphe nuclei, the periaqueductal gray, the red nucleus, the Edinger-Westphal nucleus and the interstitial nucleus of Cajal. In the diencephalon, they were observed in the dorsal thalamus and the hypothalamic regions. In the caudal medulla, labeled fibers and their terminals were observed in the nucleus prepositus hypoglossi, the nucleus intercalatus and the inferior olive. Descending axons from the NRGc coursed bilaterally through the ventral and ventrolateral funiculi as far caudal as the upper thoracic cord. Single axon collaterals arising from the descending axons gave off terminal fibers to the left or the right gray matter. Their terminals were located in laminae V-X, mainly in laminae VII and VIII. In lamina IX, they were distributed mainly in the medial portion. A few fibers originating from the descending axons ipsilateral to the PHA-L injection side coursed through the anterior or posterior commissure, and ended in laminae VI, VII and VIII. The functional implications of these findings are discussed in relation to the behavioral state control and the generalized motor inhibition.

Afferent Pathways↗

Evidence for interconnections between the two segments of the globus pallidus in primates: a PHA-L anterograde tracing study.

Small injections of the lectin Phaseolus vulgaris-leucoagglutinin (PHA-L) in the external segment of the pallidum (GPe) in the squirrel monkey (Saimiri sciureus) and in the rhesus monkey (Macaca mulatta) led to anterograde labeling of fibers in the internal segment of the pallidum (GPi). These fibers formed numerous large varicosities reminiscent of terminal boutons, which closely surrounded GPi cell bodies and primary dendrites. Conversely, PHA-L injections in the GPi of squirrel monkeys produced anterograde fiber labeling in the GPe. However, in contrast to fibers in GPi, those in GPe did not make prominent pericellular contacts. Instead, they displayed a rather linear course, had long intervaricose segments, and appeared to contact several GPe neurons along their course by close appositions on cell bodies and primary dendrites. These results suggest the existence of a reciprocal connection between the two pallidal segments, which may play a crucial role in the functional organization of the basal ganglia in primates.

Animals↗

A direct neuronal projection from the entopeduncular nucleus to the globus pallidus. A PHA-L anterograde tracing study in the rat.

The efferent projections of the rat entopeduncular nucleus were examined by use of the anterogradely transported lectin Phaseolus vulgaris-leucoagglutinin (PHA-L). Injections of PHA-L into different parts of the entopeduncular nucleus resulted in a moderate number of labeled nerve fibers in the ipsilateral globus pallidus. The fibers displayed a heterogeneous morphology: some were of small caliber with few delicate varicosities, others were of medium caliber with several more bulbous nerve terminals. Restricted injections in the dorsal and ventral parts of the entopeduncular nucleus, respectively, showed that the dorsal part of the entopeduncular nucleus projects to the dorsal and rostral parts of the dorsal pallidum and the ventral part to the ventral and caudal parts.

Animals↗

Efferent projections from the region of the medial zona incerta containing A13 dopaminergic neurons: a PHA-L anterograde tract-tracing study in the rat.

Potential efferent projections of A13 dopaminergic (DA) neurons were identified in the present study by examining the distribution of labelled fibers following iontophoretic injection of the anterogradely transported lectin Phaseolus vulgaris leucoagglutinin (PHA-L) into the medial zona incerta (MZI), the region of the diencephalon containing A13 DA neuronal perikarya. One week after injection, PHA-L labelled fibers were found throughout the brain with the heaviest labelling occurring ipsilateral to the injection site in the anterior hypothalamic area, lateral hypothalamus, lateral preoptic area, horizontal diagonal band of Broca, and parvocellular region of the paraventricular nucleus. Moderate labelling was observed in the ipsilateral median preoptic nucleus, lateral septum, lateral aspect of the bed nucleus of the stria terminalis, and central nucleus of the amygdala. Moderate labelling was also found in the contralateral MZI and parvocellular region of the paraventricular nucleus. Light labelling was detected in the ipsilateral medial preoptic area, supraoptic nucleus, ventromedial nucleus, arcuate nucleus, vertical limb of the diagonal band of Broca, and in the contralateral lateral hypothalamus. Few immunopositive fibers were present in the dorsomedial nucleus of the hypothalamus or the magnocellular region of the paraventricular nucleus. These results reveal that neurons located in the MZI (possibly A13 DA neurons) have ipsilateral efferent axonal projections to a variety of brain regions including the lateral hypothalamus, lateral preoptic area, and the limbic structures at the diencephalic-telencephalic juncture.

Animals↗

Age-related changes in LFA-1 expression, cell adhesion, and PHA-induced proliferation by lymphocytes from senescence-accelerated mouse (SAM)-P/8 and SAM-R/1 substrains.

Accelerated senescence-prone mice of the SAM-P/8jf series were compared with senescence-resistant SAM-R/1 controls in terms of age-related changes in phytohemagglutinin (PHA) proliferative responses and lymphocyte function-associated antigen-1 (LFA-1) utilization by non-adherent splenocytes. Advancing age was associated with a reduction in cell proliferative responses to PHA in both substrains, although the rate of decline was significantly more rapid in the senescence-prone animals. Conversely, in both substrains there was a progressive age-related increase in the proportion of splenocytes expressing high levels of LFA-1, and a parallel increase in the degree of LFA-1-dependent cell aggregation induced by phorbol ester. Age-matched SAM-P/8jf and SAM-R/1 mice did not differ in terms of LFA-1 expression or LFA-1-dependent cell aggregation. Two-color cytofluorometric analysis demonstrated the enhanced expression of LFA-1 expression by cells bearing the Pgp-1hi phenotype characteristic of memory lymphocytes. These results suggest that age-associated changes in lymphocyte adhesion are attributable to alterations in the relative numbers of memory cells expressing high levels of LFA-1, but are unlikely to contribute to the reduced proliferative response to mitogen in aged mice.

Aging↗

In vitro culture of lymphocyte colonies in agar capillary tubes after PHA-stimulation.

Human peripheral lymphocytes were stimulated with phytohemagglutinin (PHA) in liquid culture, suspended in agar and incubated in glass capillary tubes. Compact colonies of lymphocytes were found growing along the tube bottom in a buffer film, while compact clusters and rare diffuse colonies were observed inside the agar. Several parameters affecting the clonal growth were studied and optimized: PHA-dose, agar concentration, gel length (volume), quantity and density of seeded cells per capillary and gel length. Colony yield mainly depends on the seeded-cell density with a sharp optimum at 2 X 10(5) cells/ml irrespective of gel length; higher cell densities reduce the colony yield, suggesting that colony growth is the result of both stimulatory and inhibitory factors produced by cooperating cells. Following the daily clonal growth was only possible with undisturbed tubes; the number of colonies steadily increased from day 2 until day 7. Densitometric colony scanning is possible, yet problematic. Colony yield (plating efficiency is 10--50-fold higher in agar capillaries than in the usual Petri dishes. An additional advantage is that the capillaries provide a basis for a simple and reliable assay system for determining regulatory factors of lymphocyte proliferation (including chalones).

Agar↗

A sensitive method to measure PHA-induced cytotoxicity to adherent target cells using [3H]uridine as a terminal label.

This paper describes a modification of the PHA-induced cytotoxicity test of human peripheral blood lymphocytes against a tumour-derived adherent cell line (HeLa), in which the surviving target cells are labelled with [3H]uridine at the end of the assay. There is a direct correlation between [3H]uridine incorporation and the number of adherent target cells. The test proves to be very sensitive at low effector; target cell ratios. Frozen stored cells can be used in this system, a particular advantage because of the possibility of increasing the reproducibility of the assay by using the same batch of cryopreserved lymphocytes as a reference standard in each experiment. PHA-induced cytotoxicity was mainly found in the T cell enriched fraction.

Adolescent↗

Different optimal PHA concentrations for stimulation of Chinese hamster lymphocytes in cultures supplemented with foetal calf serum or horse serum.

Chinese hamster lymphocyte cultures were supplemented with foetal calf serum (FCS) or hose serum (HS). Addition of 2-mercaptoethanol (2-ME) to cultures supplemented with these sera resulted in a marked increase lymphocyte proliferation. The PHA concentrations necessary for optimal lymphocyte stimulation were much lower in cultures supplemented with HS than with FCS. Phytohaemagglutinin (PHA) concentrations optimal for stimulation of a given number of lymphocytes in cultures supplemented with FCS often gave inhibition of similar cultures supplemented with HS.

Animals↗

Activation by PHA of CD8 lymphocytes into clonal colony forming cells. Role of interleukin-1.

Monoclonal T cell colonies can be grown in agar culture from quiescent T lymphocytes under PHA stimulation, provided that (1) a low number of T lymphocytes (less than or equal to 5 X 10(4)/ml) is seeded, (2) IL-2 is added to the culture, and (3) a high number of accessory B cells (greater than or equal to 5 X 10(5)/ml) is present in contact with the T lymphocytes. Under these culture conditions the colony progenitors can be ascribed to the CD4 subset, whereas CD8 lymphocytes do not generate colonies. This finding is surprising since both CD4 and CD8 lymphocytes may be cloned in liquid culture. We now report the appropriate conditions required to grow cytotoxic CD8 lymphocyte colonies in agar. CD8 colony growth is dependent upon IL-2-IL-2 receptor interaction and is inhibited by anti-IL-2 receptor antibodies. In addition to PHA, accessory B cells and IL-2, an additional signal provided by recombinant IL-1 is necessary for CD8 colony formation. Exogenous IL-1 can be replaced by irradiated CD4 lymphocytes which stimulate the expression of membrane IL-1 activity in the accessory B cells. In addition, colony growth from quiescent but not preactivated CD8 lymphocytes is inhibited by anti-IL-1 antibodies. Altogether, the data show that an IL-1 signal is required for the induction of IL-2 responsive IL-2 receptors on quiescent CD8 colony forming cells.

Animals↗

Survival and PHA-stimulation of gamma-irradiated human peripheral blood T lymphocyte subpopulations.

Human peripheral blood T lymphocyte subpopulations were identified and isolated on the basis of their ability to bind IgG (T-G), IgM (T-M), or neither immunoglobulin class (T-null). Lymphocytes were exposed to 0, 0.5, 1.0, 2.5 or 5.0 Gy of 60Co gamma-rays either as a T-cell suspension or as separated T cell subsets. Survival curves, determined 5 days after irradiation, revealed that each subset has radiosensitive and radioresistant portions, and that the T-G cell is the most sensitive subset. Mitotic indices of 48-h cultures showed that the response of unirradiated T lymphocytes to PHA varied greatly among the subsets, the highest indices being obtained for the T-M and the lowest for the T-G cells. With the possible exception of the T-G cells, the subsets are relatively resistant to mitotic effects of gamma-rays. T-G cells suppress the PHA-induced mitotic response of the other T lymphocyte subsets, and this suppressor effect is radiosensitive, being abolished by 1.0 Gy. It is concluded that lymphocytes exposed to greater than or equal to 1 Gy of gamma-rays will have very few dividing B lymphocytes or T-G cells. This together with radiation-induced loss of T-G suppressor action means that the predominant lymphocyte types in mitosis after greater than or equal to 1 Gy are the radioresistant T-M and T-null cells.

Cell Survival↗