Augmentation of antiproliferative and antitumor effect on human cancer cells in combined use of electroporation with a plant toxin, saporin.

Effect of electroporation (EP) in combination with a plant toxin, saporin, was studied using a human lung cancer cell line (PC9) and a pancreatic cancer cell line (ASPC-1). Target cells were electroporated in the presence of saporin and washed, and incubated for 72 hr. Proliferation inhibition in combination of EP and saporin was observed in parallel with the voltages and the saporin concentrations used. Proliferation of PC9 cells was completely inhibited at 1000 ng/ml of saporin in combination with EP (80-90 V, 10 ms, n = 8). High degree of proliferation inhibition was also obtained when ASPC-1 cells were electroporated in the presence of saporin (0.1-1000 ng/ml). PC9 or ASPC-1 tumor-bearing nude mice were treated with electroporation following the intratumoral injection of saporin (1 mg). Tumor necrosis was observed 24-48 hr after the combination therapy with saporin and EP. Six of nine mice with established PC 9 tumors and all the mice with established ASPC-1 tumors regressed completely 14 days and 6 days after the combination therapy, respectively.

Hypoplastic basement membrane of the lens anlage in the inheritable lens aplastic mouse (lap mouse).

Adult homozygous lap mice show various eye abnormalities such as aphakia, retinal disorganization, and dysplasia of the cornea and anterior chamber. In the fetal eye of a homozygous lap mouse, the lens placode appears to develop normally. However, the lens vesicle develops abnormally to form a mass of cells without a cavity, and the mass vanishes soon afterward. Apoptotic cell death is associated with the disappearance of the lens anlage. We examined the basement membranes of the lens anlage of this mutant by immunohistochemical methods under light microscopy using antibodies against basement membrane components of the lens anlage, type IV collagen, fibronectin, laminin, heparan sulfate proteoglycan, and entactin and by transmission electron microscopy. Immunohistochemistry showed the distribution and intensity of antibody binding to the lens anlage to be almost the same for each these antibodies regardless of the stage of gestation or whether the anlagen were from normal BALB/c or lap mice. Thus, positive continuous reactions were observed around the exterior region of the lens anlage from day 10 of gestation for type IV collagen, fibronectin, laminin, heparan sulfate proteoglycan antibodies, and at least from day 11of gestation for entactin antibody. The basement membrane lamina densa of both normal and lap mice was shown by electron microscopy to be discontinuous at days 10 and 10.5 of gestation. However, by day 11 the lamina densa was continuous in the lens anlagen of normal mice but still discontinuous in the lap mice. By day 12 of gestation, the lamina densa had thickened markedly in normal mice, whereas in lap mice it remained discontinuous and its thinness indicated hypoplasia. These results indicate that, while all basement components examined are produced and deposited in the normal region of the lens anlage in the lap mouse, the basement membrane is, for some reason, imperfectly formed. The time at which hypoplasia of the basement membrane was observed in this mutant coincided with the stage during which apoptosis in the lens anlage occurred. This result may indicate a possibility of the relationship between the basement membrane and apoptosis in this mutant.

[The crystallinity of hypomineralized rat enamel caused by fluoride administration].

It has been well known that there are extreme amounts of inter-crystalline spaces and large amounts of enamel matrix in fluorosed human enamel. Our previous study discussed the fact that amelogenins in developing fetal enamel matrix protein may have a role in regulating or controlling enamel crystal growth. These results positively suggest that amelogenins degradation may be associated with the etiology of fluorosed enamel. The purpose of this study was to examine the relationship of the crystallinity of fluorosed enamel of rats and the molecular weight of enamel matrix protein. The crystallinity of fluorosed enamel of rats, caused by the ingestion of fluoride containing water was evaluated with the microbeam x-ray diffraction analysis and the molecular weight of enamel matrix protein was examined by SDS-polyacrylamide-gel electrophoresis. The results obtained in the study are summarized as follows. 1) The plasma fluoride level of rats increased linearly with the fluoride concentration in the drinking water. 2) The microradiographs of maturing incisor enamel of the group with 100 ppmF- and 200 ppmF- injected showed diffused radiolucent zone from the subsurface toward the dento-enamel junction. The disturbances in enamel mineralization were most apparent in the 200 ppmF- group. 3) The crystallinity of the radiolucent zone of the fluorosed enamel decreased in both the a and c-axis directions, and particularly decreased more in the a-axis compared with the control. 4) The pattern of the SDS-polyacrylamide gel electrophoresis of the enamel protein in the mature stage showed higher remaining molecular weight of amelogenins in rats in the 100 ppmF- and 200 ppmF- group. These results suggest that in hypomineralized enamel caused by long-term administration of fluoride containing water, the degradation of amelogenin protein was disturbed, and consequently the crystallinity of enamel apatite decreased.

[Development of chewing center at transition period from sucking to chewing].

UNLABELLED: The purpose of this study was to identify and to characterize the peripheral movement evoked by electrical stimulation given to the masticatory area of the cerebral cortex (Control group), and to clarify the development of the chewing center by examining the change in the peripheral movement when the tooth germs have been enucleated (Enucleation group). Puppies were used in the present study which were anesthetized with a 25% solution of Urethane (4 ml/kg, i.p.). Their heads were then, fixed in a stereotaxic apparatus, and the surfaces of the orbital gyrus was surgically exposed. Electrical stimulation (Frequency: 25 c/sec, DURATION: 2 msec) was given to the masticatory area. All of the tooth germs of the deciduous teeth were enucleated at 12-14 days of age in the enucleation group. After the enucleated wound healed, stimulation was similarly performed in the control. The results obtained were as follows; In the control group, a sucking movement was evoked coinciding with the beginning of the eruption of the deciduous teeth (19-23 days of age), and it changed into a chewing movement as eruption proceeded. However, by 14 days of age, neither sucking nor chewing movements were evoked even when stimulation was applied to the area of the cerebral cortex. In the enucleation group, the time of the onset of the sucking movement was considerably delayed until the age of 22-27 days. Also it was converted into a jaw opening movement which occurred after 28 days of age. The chewing movement was observed for some days after 41 days of age. The transition from sucking to chewing was recognized to undergo a little delay when the deciduous teeth was enucleated. These results suggest the afferent impulses provided with the eruption of deciduous teeth presumably play an important role on the development of the chewing center and consequently on the transition from sucking to chewing.