Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

New PCR-RFLP analysis for the mouse Tnfsf6gld gene caused by a point mutation in the Tnfsf6 [tumor necrosis factor (ligand) superfamily, member 6] locus.

A new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of the mouse Tnfsf6gld mutation. An artificial restriction site was introduced to the mouse Tnfsf6gld mutation by PCR amplification using a modified primer. The three genotypes of the Tnfsf6 locus (Tnfsf6gld/Tnfsf6gld, Tnfsf6gld/+, and +Tnfsf6-gld/+Tnfsf6-gld) could be distinguished clearly and easily. This PCR-RFLP analysis was found to be useful for the identification of the mouse Tnfsf6gld mutation.     

Scale Arrangement Pattern in a Lepidopteran Wing. 1. Periodic Cellular Pattern in the Pupal Wing of Pieris rapae

In most species of lepidopteran insects, anteroposterior rows formed by scales are arranged at regular intervals in the adult wing; within each row two kinds of scales are alternately arranged. To investigate the cellular basis for the scale arrangement pattern, we examined cell arrangement in the epidermal monolayer of the pupal wing of a small white cabbage butterfly, Pieris rapae , by scanning electron microscopy and light microscopy.
The arrangement of scale precursor cells, closely resembling that of scales in the adult wing, was observed in the wing epidermis of the early pupa. Scale precursor cells are proximodistally elongated and form anteroposterior rows. Within a row two kinds of scale precursor cells are nearly alternately arranged, which is not so precise as the alternation of scales in the adult wing. Individual rows of scale precursor cells are separated by rows of single or double undifferentiated general epidermal cells. Occasionally, arrangement abnormalities occur both in the adult and the pupal wing. The cellular basis for the regular spacing of scale rows is discussed.     

Decrease in CuZn-superoxide dismutase mRNA level during differentiation of human monocytic and promyelotic leukemia cells

Change in the level of CuZn-superoxide dismutase (SOD) mRNA was examined using a molecular probe during differentiation of human monocytic leukemia U937 cells or promyelotic leukemia HL-60 cells induced by either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or dimethylsulfoxide (DMSO). CuZn-SOD mRNA levels were found to decrease during the course of differentiation, and this response is specific for differentiation, since the treatment of human B cell leukemia cells or normal diploid fibroblasts with TPA failed to have any effect on the level of CuZn-SOD mRNA. The activity of CuZn-SOD in U937 cells also decreased during differentiation, but following that of the CuZn-SOD mRNA level. The expression of the CuZn-SOD gene is thus concluded to diminish during the differentiation of HL-60 and U937 cells.     

Changes of DNA methylation level during pre-and postnatal periods in mice

DNA methylation in an adult mammalian body shows tissue-specificity. But when and how the specificity is established in the process of development has not yet been elucidated. Here we have investigated age-dependent changes in the amount of 5-methyldeoxycytidine (5mdC) that DNA of various mouse tissues contains during the late-fetal and postnatal periods, using high-performance liquid chromatography. The tissue-specificity in the 5mdC level was observed in the late-fetal stage, and the level continued to change during the subsequent periods. The most pronounced alterations were observed in brain and liver, where similar biphasic changes were seen, but at different ages. At maturation, the 5mdC levels were high in thymus, spleen and brain, intermediate in lung, and low in liver and sperm. The data demonstrate the importance of the peri- and postnatal periods in establishment of tissue-specificity in 5mdC content.     

Role of cardiopulmonary baroreflexes during dynamic exercise

To examine the role of cardiopulmonary (CP) mechanoreceptors in the regulation of arterial blood pressure during dynamic exercise in humans, we measured mean arterial pressure (MAP), cardiac output (Q), and forearm blood flow (FBF) during mild cycle ergometer exercise (77 W) in 14 volunteers in the supine position with and without lower-body negative pressure (LBNP). During exercise, MAP averaged 103 +/- 2 mmHg and was not altered by LBNP (-10, -20, or -40 mmHg). Steady-state Q during exercise was reduced from 10.2 +/- 0.5 to 9.2 +/- 0.5 l/min (P less than 0.05) by application of -10 mmHg LBNP, whereas heart rate (97 +/- 3 beats/min) was unchanged. MAP was maintained during -10 mmHg LBNP by an increase in total systemic vascular resistance (TSVR) from 10.3 +/- 0.5 to 11.4 +/- 0.6 U and forearm vascular resistance (FVR) from 17.5 +/- 1.9 to 23.3 +/- 2.6 U. The absence of a reflex tachycardia or reduction in arterial pulse pressure during -10 mmHg LBNP supports the hypothesis that the increase in TSVR and FVR results primarily from the unloading of CP mechanoreceptors. Because CP mechanoreceptor unloading during exercise stimulates reflex circulatory adjustments that act to defend the elevated MAP, we conclude that the elevation in MAP during exercise is regulated and not merely the consequence of differential changes in Q and TSVR. In addition, a major portion of the reduction in FBF in our experimental conditions occurs in the cutaneous circulation. As such, these data support the hypothesis that CP baroreflex control of cutaneous vasomotor tone is preserved during mild dynamic exercise.     

Difference in rehydration process due to salt concentration of drinking water in rats

Albino rats were thermally dehydrated (approximately 8% of body wt), divided into five groups, and given tap water or 0.2, 0.45, 0.9, or 2.0% NaCl solution ad libitum for 16 h. Rats given 0.9 or 0.45% NaCl solution regained fluid loss completely in 3-3.5 h, whereas those given 0.2% solution became fully rehydrated at 10 h. The rats in the tap water and 2.0% NaCl groups were only 78 and 59% rehydrated, respectively, within 16 h. Na balance was positive in the 0.9% NaCl group by about five times the amount of the cations lost during the dehydration period. A positive balance of Na was also observed in the 0.45 (approximately 250%) and 2.0% NaCl groups (300%), whereas the 0.2% NaCl group regained lost water and Na simultaneously at 10 h. With tap water, additional loss of cations was observed. These findings show that for the replacement of water due to thermal dehydration there is a range of NaCl concentration with which the rats can rehydrate with the mutual cooperation of thirst, salt appetite, and kidney function.     

Evidence for the location of foreign genes in three different chromosomes in a plant obtained from direct DNA transfer

Tobacco mesophyll protoplasts were treated with plasmids, pCT2 (17.1 kbp) or pCT2T3 (18.3 kbp), which contained a chimeric aminoglycoside phosphotransferase II (APH(3′)II) gene and an intact nopaline synthase gene. Expression of two marker enzymes, APH(3′)II and nopaline synthase, were analyzed in transformed plants. Four out of 16 transformants obtained by pCT2T3 possessed both enzymes. Upon self-pollination, the progeny of one of transformants (T2) segregated to 153∶4 in terms of resistant and susceptible character to kanamycin, suggesting insertion of foreign genes into three independent chromosomes. The kanamycin resistant character in the rest of transformants showed 3∶1 segregation. DNA blot analysis of the T2 transformant and progenies indicated the presence of two marker genes.     

The combined effect of lymphokine activated killer cell and radiation therapy on rat brain tumorin vitro

Thein vitro effect of a combined treatment with lymphokine activated killer (LAK) cell and radiation therapy on rat brain tumor was examined using⁵¹Cr release assay. The tumor cell-line used in this experiment was 9L rat brain tumor derived from a Fischer 344 rat. LAK cells were obtained by culturing rat lymphocytes with recombinant human interleukin 2 for at least 3 days. The cytotoxic activity of the LAK cells was examined by⁵¹Cr release assay. Irradiation was done by exposing the microtiter plate in which the¹⁵Cr labeled 9L cells and LAK cells were cultured to a¹³⁷Cs gamma cell unit. Without irradiation, there was 18% cytotoxicity in the 1:100 tumor-to-LAK cell ratio specimen after 24 hrs cocultivation. However, if 5 Gy of irradiation was given, followed by 12 hrs incubation, the cytotoxicity was enhanced significantly at the same cell ratio (30%). This enhancement effect was the most prominent when the cell ratio was 1:100 and the irradiation dose was 5 Gy. To generate the enhancement effect, an incubation time of over 8 hrs both before and after irradiation was required. The supernatant of the LAK cells showed 19.8% and 11.4% cytotoxicity with and without irradiation, respectively. This result indicates the participation of a cytotoxic factor released from LAK cells.This work is supported in part by grant from Univeristy of Tsukuba Project Research.