Junko Nishiitsutsuji-Uwo

Mode of action of Bacillus thuringiensis δ-endotoxin: Histopathological changes in the silkworm midgut

Abstract Quite different ultrastructural changes were observed in the columnar cell and the goblet cell of the silkworm midgut after administration of the crystalline toxin of Bacillus thuringiensis . Shortly after the ingestion of the toxin, the deep infoldings of the basal cell membrane of some columnar cells became very irregular in shape and the mitochondria near the basal region were transformed into a condensed form. A few goblet cells showed relatively high electron density in the cytoplasm. The earliest pathological changes were slight and located in a region lying between the first and second thirds of the midgut. With the passage of time, they spread anteriorly and posteriorly to include the entire anterior two thirds of the midgut and became more profound. The cytoplasm of columnar cells became very electron transparent. Most mitochondria were transformed into a condensed form and the endoplasmic reticulum assumed a vacuole-like configuration. The basal infoldings of the cell membrane almost disappeared. On the other hand, the cytoplasm of the goblet cells became very electron dense and granular. The clear basal infoldings of the cell membrane were enlarged making a striking contrast with the dense cytoplasm. However, the mitochondria and the endoplasmic reticulum did not show any pathological deformation.

Evolutionary aspects of “brain-gut peptides”: An immunohistochemical study

Phylogeny of biogenic peptides and their source cells was studied by immunohistochemistry and electron microscopy. The distribution of the peptide containing neurons and paraneurons in the brain and in the gastroenteropancreatic endocrine system was depicted, especially in the bullfrog as the representative of deuterostomia and in the cockroach and some other insects as the representatives of protostomia. Stress was given to: (1) calcitonin-immunoreactive neurons in bullfrog hypothalamus and PP-reactive neurons in the cockroach protocerebrum as instances of transmissional-hormonal partition of a neuropeptide, (2) open-type endocrine cells in the gut structurally and functionally common to the protostomia and deuterostomia, and (3) phylogeny of the prohormones with special reference to big gastrin and proglucagon (glicentin).

Localization of pancreatic polypeptide (PP)-like immunoreactivity in the central and visceral nervous systems of the cockroach Periplaneta

SummaryThe central and visceral nervous systems of the cockroach Periplaneta americana were studied by means of the peroxidase-antiperoxidase immunocytochemical method, with the use of antibody to bovine pancreatic polypeptide (PP). PP-like immunoreactive neuron somata are most numerous in the brain; at least 6 pairs of cell groups occur in clearly defined regions. Three pairs of cells each are also present in the suboesophageal ganglion and the thoracic ganglia, one pair of a single cell each in the first abdominal and the frontal ganglia, and 4 to 6 pairs of single cells in the terminal ganglion. No reactive cells were found in the retrocerebral complex and the second to the fifth abdominal ganglia. The axons containing PP-like immunoreactivity issue many branches that are distributed in the entire brain-retrocerebral complex, ventral cord, and visceral nervous system. PP-like immunoreactive material produced in the brain seems to be transported by three routes: protocerebrum to corpora cardiaca (-allata) through the nervi corporis cardiaci, tritocerebrum to visceral nervous system through frontal commissures, and to ventral cord through circumoesophageal connectives.A possible homology between the mammalian brain-GEP (gastro-enteropancreatic) system and the brain-midgut system of this insect is discussed.

Mode of action of Bacillus thuringiensis δ-endotoxin: Effect on TN-368 cells

Abstract TN-368 cells swelled and burst upon treatment with the dissolved δ-endotoxin of Bacillus thuringiensis . However, the cytotoxic response was greatly affected by the ionic conditions of the solutions employed for the toxin tests. Ions, in addition to K + , seemed to participate in the cytotoxic expression of δ-endotoxin, if their concentration was sufficient (>100 m m ). Although similar swollen cells were observed with valinomycin treatment, some differences appeared on the ultrastructural level, especially in the mitochondria. Those of the toxin-treated cells were transformed into the “condensed” form, while those of valinomycin-treated cells were transformed into the “swollen” form. Therefore, the cytotoxic effect of δ-endotoxin, unlike that of valinomycin, seemed to be a general breakdown of ion regulation on the cell level.

Exocytotic release of secretory granules from endocrine cells in the midgut of insects

SummaryExocytotic release of the secretory granules of the endocrine cells in the midgut of a cockroach, Periplaneta americana, was studied by means of fixation with tannic acid in combination with glutaraldehyde and osmium tetroxide. A sequence of images indicative of exocytosis suggests the following steps in this process: (1) A delicate connection appears between the granule-limiting membrane and the plasma membrane. (2) The plasma membrane approaches the granule, forming a concave indentation. (3) The granule-limiting membrane fuses with the plasma membrane and opens to give rise to an omega profile. (4) The granule content is voided into extracellular space. Exocytosis occurs not only at the base of the cell but occasionally at its side facing adjacent cells. (5) The exocytotic invagination after release becomes smaller and narrower; sometimes a coated pit with bristles appears. Multiple exocytosis, and exocytosis in the endocrine cells of the nidus, i.e., the regenerative cell mass, are also described.

Factors affecting the insecticidal activity of δ-endotoxin of Bacillus thuringiensis

Abstract The stability and solubility of the crystal toxin of Bacillus thuringiensis var. aizawai were measured using larvae of Bombyx mori as the test animals under the same test conditions. Insecticidal activity was retained in buffer solutions of pH 2.2–10.8. It decreased rapidly either below pH 2.0 or above pH 11. As to the effects of time and temperature, the longer the incubation time, the lower was the activity in solutions above pH 11, and the higher the treatment temperature, the lower was the pH at which degradation of activity occurred. Guanidine (6 m ) with 1% mercaptoethanol was not good for dissolving the toxic protein. Instead, 8 m urea with 0.5% dithiothreitol or with 1% mercaptoethanol and 0.04 m NaOH seemed better, and onefourth of the total activity appeared in the soluble fraction. On the other hand, 0.2 m thioglycollate and “Elliss” buffer dissolved the toxic moiety completely and safely.

Mode of action of Bacillus thuringiensis δ-endotoxin: General characteristics of intoxicated Bombyx larvae

Abstract The general pathology induced by δ-endotoxin in terms of larval behavior and hemolymph chemistry has been widely studied in the so-called Type I insect, Bombyx mori. The succession of symptoms is divided into four arbitrary stages: Stage 0, appearance and locomotion normal, no feeding; Stage 1, slightly sluggish; Stage 2, extremely sluggish; and Stage 3, complete paralysis. The action of δ-endotoxin is highly specific to the midgut since contractile movement of both foregut and hindgut continues long after all locomotor activity and heartbeat have stopped. Immediately after the silkworm stops feeding and blood pH sharply rises, there is an associated abrupt rise in the K+ concentration of hemolymph. Thereafter, the rise in K+ is linear while the rise in pH is not. In vivo measurements have not yielded the same simple linear dependence of pH on K+ concentrations that is found in in vitro mixtures of hemolymph and midgut juice. Ligation experiments showed that the same pathological sequence (rise of pH and K+ concentration, and general paralysis) follows whether the toxin has unrestricted access to the entire midgut or only part of it (anterior or posterior). From the results of injections of midgut juice or various salt solutions into hemocoel, we came to the conclusions that the blood pH and the symptoms are not necessarily parallel and the intact midgut and Malpighian tubules have strong functions for ion regulation.

Partial purification and properties of prothoracicotropic hormone (PTTH) from developing adult brains of the silkworm Bombyx mori

Abstract The extraction and purification of prothoracicotropic hormone (PTTH) from brains of the developing adults of Bombyx mori are described. One brain contained about 40 Samia units of PTTH. Sixty-five thousand brains were processed through a nine-step procedure that included heat treatment, phenol extraction, repeated gel filtration and ion-exchange chromatography to produce a highly active preparation in 56% yield, which initiated adult development of a debrained Samia pupa at a dose of 4 ng. Although the purification was achieved only by 683-fold at the step 9, the purity of the PTTH preparation seemed to correspond to a 43,000-fold purification of the PTTH preparation when started from the whole adult head. The mol. wt of the PTTH was estimated to be 5000 or less and the isoelectric point was approx. pH 4.4. The PTTH preparation was inactivated by the treatment of proteases such as Nagarse, pepsin and thermolysin, indicating its peptidal nature.

Sporeless mutants of Bacillus thuringiensis. III. The process of crystal formation

In order to investigate the formation of the parasporal crystal of B. thuringiensis with special reference to the spore, sequential ultrastructural analysis of sporulation was performed using a sporeless mutant strain (sp-) as well as its parent wild strain (sp+). From the logarithmic growth to the end of forespore formation, the same sequential process of sporulation proceeded in both strains and a forespore with double membranes appeared. Thereafter, subsequent sporulation in the sp- strain was either partly or completely arrested and finally spore (mainly the forespore) became deformed. On the other hand, crystal formation took place throughout by the same processes both in sp+ and sp- strains. During the forespore formation, a primordial crystal and an ovoid inclusion appeared and after this stage, the crystal displayed a characteristic diamond-shaped body with lattice fringes increasing its size. No regularity was found in the position of the crystal with respect to the spore. As far as the present ultrastructural observations were concerned, the crystal developed without any special association with the membranes of the spore. However, without the formation of the forespore (including the incipient forespore), no crystal formtion was observed.