Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)     

Identification of neuroendocrine cells producing a diuretic hormone in the tobacco hornworm moth,Manduca sexta

Summary Separate antisera were raised to the N- and C-terminal half of the diuretic hormone from Manduca sexta. Antisera against the two halves of this peptide recognized the same cells in M. sexta, and preabsorption of the antisera with the peptides used as antigens abolished the immunoreactivity, confirming their specificity. The antisera reacted with two median neurosecretory cells on each side of the protocerebral groove in larvae, and with a group of about 80 small median neurosecretory cells in the adult, as well as their axons to, and their axon terminals in, the corpora cardiaca. During the early pupal stages, small cells, which are possibly derived from a common neuroblast, differentiate into immunoreactive neurosecretory cells, which explains the large increase in cell numbers in the adult. In the sleepy sulphur butterfly, Eurema nicippe, homologous median neurosecretory cells in the adult were immunoreactive with both antisera.     

Population differences in larval hostplant use in the checkerspot butterfly, Euphydryas chalcedona

Larvae from two populations of Euphydryas chalcedona Doubleday & Hewitson (Nymphalidae) were reared on their own hostplant and that of the other population, in both pre-diapause and post-diapause instars. One population, Chico, uses Penstemon breviflorus Lindl. (Scrophulariaceae), and the other, Echo Lake, uses P. newberryi Gray. Growth rate and survival were determined for pre-diapause and post-diapause larvae from both populations on both plant species; and digestive efficiencies were calculated during the prediapause instars. The results showed that larvae from the two populations differed in their responses to the two plant species. Pre-diapause larvae from Chico performed equally well on both plant species—survival and digestive indices were not significantly different for two Penstemon species. In contrast, pre-diapause larvae from Echo Lake performed significantly worse on the non-hostplant—growth and survival were significantly lower on the non-host, P. breviflorus. In addition, comparison of digestive efficiencies for the two plants showed that larvae from Echo Lake digested P. breviflorus better than P. newberryi, but were significantly less able to convert P. breviflorus to body mass. In the post-diapause instars, larvae from Chico grew faster on the host than on the non-host. Larvae from Echo Lake grew quite slowly on both plant species and significantly more of the Echo Lake larvae returned to diapause instead of completing development.

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Résumé Des chenilles de deux populations d'E. chalcedona ont été élevées sur leur propre plante-hôte et sur celle de l'autre population, aux stades avant et après diapause. Les deux populations s'alimentent sur différentes espèces de Penstemon (Scrophulariaceae), et une population—Echo Lake—est monophage sur P. newberry, tandis que l'autre—Chico—utilise d'abord P. breviflorus, mais les chenilles après diapause sont trouvées sur au moins deux autres espèces de plantes. Les taux de croissance et de survie ont été déterminés pour des chenilles avant et après diapause pour les deux populations sur les deux plantes; les efficacités digestives ont été calculées sur les chenilles avant diapause.Les résultats ont montré que les chenilles des deux populations différaient par leur degré de spécialisation digestive sur leur plante hôte normale: les chenilles de Chico ont utilisé aussi bien les deux plantes, tandis que celles d'Echo Lake le faisaient significativement moins bien sur la plante non-hôte, par suite de l'inaptitude à la digérer. Ainsi la population oligophage est alimentairement moins spécialisée et plus capable de se débrouiller avec une plante non-hôte. Après diapause, les chenilles de Chico s'alimentaient significativement mieux sur plante hôte que non-hôte, ce qui était le cas aussi pour la population monophage. Dans l'ensemble, les chenilles de la population monophage semblaient moins capables de se débrouiller dans des conditions défavorables ou moins avantageuses.

     

Biology of Subanguina picridis,a Potential Biological Control Agent of Russian Knapweed

The knapweed nematode, Subanguina picridis, forms galls on the leaves, stems, and root collar of Russian knapweed, Acroptilon repens. After being revived from a dormant, cryptobiotic state, second-stage juveniles required at least 1 month in a free-living state before becoming infective. Galls were induced on relatively slow-growing host plants that retained their apical meristems at or near the soil surface for 2-5 weeks. Galls developed extensive areas of nutritive tissue. The nematode was introduced from the Soviet Union and released in Canada for the biological control of Russian knapweed.     

THE HOST RANGE OF THE CHLORELLAVOROUS BACTERIUM ("VAMPIROVIBRIO CHLORELLVORUS")

The chlorellavorous bacterium . "Vampirovibrio chlorellavorus" Gromov et Mamkaeva, grows only by killing and consuming the cell contents of species of the green alga Chlorella Beijerinck. Of the 76 algal strains examined in this study, the bacterium attacks all 31 strains of the species C. vulgaris. C. sorokiniana, and C. kessleri, but attacks only two of 39 strains of nine other Chlorella species. Neither of two Prototheca strains was susceptible to attack. This narrow host specificity may be related to cell surface properties .     

On the evolution of non-specific mutualism

It has been argued that mutualisms are non-specific when mutualistic interactions are weak and transient, and become more specific as interactions increase in strength. However, this runs counter to the observation that there exist tightly linked mutualisms of great antiquity that are highly nonspecific. Here we argue that mutualism generates positive, interspecific, frequency-dependent selection, which acts as a cohesive evolutionary force, discouraging evolution of specificity. A simple mathematical model is constructed to analyse the evolution of a community consisting of two guilds of species with mutualistic between-guild interactions, two competing species in each guild and two genetically distinct phenotypes within each species. With some simplifying assumptions, the trajectories in the neighbourhood of the only interior equilibrium point are determined analytically in terms of interactions between individuals. These show that the equilibrium is locally stable (no evolution) when there is little differentiation between phenotypes in mutualistic and interspecific, competitive interactions. On the other hand, when there is strong differentiation between phenotypes in their mutualistic interactions, the equilibrium is unstable and the community starts to evolve towards non-specificity. There are, however, two forces counteracting this tendency which, if sufficiently potent, cause evolution towards specificity. The first is generated by strong differentiation between phenotypes in interspecific competition; the second is caused by specificity which already exists between species in their mutualistic interactions. Thus, the tendency for non-specificity or specificity to evolve depends on the interplay between antagonistic and mutualistic interactions in the community. We illustrate these results with some numerical examples and, finally, survey some data on specificity of mutualisms in the light of the analysis.     

Mutational specificity of a proof-reading defective Escherichia coli dnaQ49 mutator

Summary The dnaQ (mutD) gene product which encodes the -subunit of the DNA polymerase III holoenzyme has a central role in controlling the fidelity of DNA replication because both mutD5 and dnaQ49 mutations severely decrease the 3–5 exonucleolytic editing capacity.It is shown in this paper that more than 95% of all anaQ49-induced base pair substitutions are transversions of the types G:C-T:A and A:T-T:A. Not only is this unusual mutational specificity precisely that observed recently for a number of potent carcinogens such as benzo(a) pyrene diolepoxide (BPDE) and aflatoxin B1 (AFB1), which are dependent on the SOS system to mutagenize bacteria, but it is also seen for the constitutively expressed SOS mutator activity in E. coli tif-1 strains as well as for the SOS mutator activity mediated gap filling of apurinic sites. Because the G:C-T:A and A:T-T:A transversions can either result from the insertion of an adenine across from apurinic sites or arise due to the incorporation of syn-adenine opposite a purine base, we postulate that the DNA polymerase III holoenzyme also has a reduced discrimination ability in a dnaQ49 background.The introduction of a lexA (Ind^-) allele, which prevents the expression of SOS functions, led to a significant reduction in the dnaQ49-caused mutator effect.Both, the mutational specificity observed and the partial lexA ⁺ dependence of the mutator effect provoke a reanalysis of the hypothesis that the DNA polymerase III holoenzyme can be converted into the postulated but until now unidentified SOS polymerase.     

Rat hypothalamic GRF elicits its biologic action in GH3 cells by interaction with VIP- preferring receptor site(s)

GH3 cells can be used effectively to study the in vitro mechanism of action of GRF. In these cells, there is a time and concentration-dependent release of cAMP into the medium. Rat hypothalamic GRF, (rGRF) is 7 to 10 fold more active than human hypothalamic GRF (hGRF). VIP, a peptide which is structurally homologous to GRF, stimulates cAMP efflux in GH3 cells, with a higher affinity than hGRF or rGRF. We propose that in contradistinction to the normal rat pituitary, the stimulation of cAMP release by GRF in GH3 cells occurs via activation of VIP-preferring receptors and that GRF (rGRF in particular) behaves as a partial VIP agonist.     

Ultrastructural analysis of pathologic lesions in sterol-deficient Nippostrongylus brasiliensis larvae

A variety of cellular lesions were manifested by the free-living larval stages of Nippostrongylus brasiliensis cultured axenically in medium lacking cholesterol. Pathologic changes developed rapidly and were most apparent in intestinal cells which displayed generalized degradation of membranous organelles. Mitochondria, endoplasmic reticulum, and Golgi complexes became disassociated and vacuolated. Autophagosomes appeared within intestinal cells and contained a wide variety of cellular components. By the 5th day gross vacuolization and degeneration of intestinal cells occurred and the hypodermis and lateral cords displayed lysed cytoplasmic regions. The latter structures are concerned with synthesis of cuticle and their degeneration correlates with the suppression of molting and the abnormal molts that occurred.