In vivo gene transfection via intravitreal injection of cationic liposome/plasmid DNA complexes in rabbits

To optimize the in vivo ocular transfection efficiency of plasmid DNA (pDNA)/cationic liposome complexes, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)/dioleoylphosphatidylethanolamine (DOPE) (1:1 molar ratio) liposomes and DOTMA/cholesterol (Chol) (1:1 molar ratio) liposomes were prepared with varying amounts of pDNA. pDNA/cationic liposome complexes were intravitreally injected (100 microL) in rabbits, and luciferase activity in the cornea, aqueous humor, iris-ciliary body, lens, vitreous body, and retina was measured. Transfection efficiency of pDNA alone did not change with pDNA ranging from 40 to 85 mg. In contrast, transfection efficiency of pDNA complexed with DOTMA/Chol liposomes significantly increased with the amount of pDNA ranging from 40 to 85 microg (P < 0.05). pDNA complexed with DOTMA/DOPE liposomes could not be prepared with pDNA greater than 60 microg. Among these experiments, pDNA (85 microg) complexed with DOTMA/Chol liposomes (pDNA:cationic liposome charge ratio (- : +) = 1.0:2.0) showed the highest transfection efficiency in the ocular tissue and its transfection-mediated luciferase activity peaked at 3 days. Among the ocular tissues, the highest gene expression was observed in the aqueous humor.     

A case of successful management of maternal septic shock with multiple organ failure following amniocentesis at midgestation

Maternal sepsis is an unusual but catastrophic complication of amniocentesis. We report a case of successful treatment of maternal septic shock and multiple organ failure following amniocentesis at midgestation, possibly due to needle puncture of the sigmoid colon, which was tightly adherent to the anterior surface of the pregnant uterus.     

Pharmacokinetics and tissue distribution of uraemic indoxyl sulphate in rats

The purpose of the present study was to examine the pharmacokinetic properties of indoxyl sulphate, a harmful uraemic toxin that accumulates during chronic renal failure. The pharmacokinetics and tissue distribution of indoxyl sulphate were examined in normal and 5/6 nephrectomized (CRF) rats. The uptake process of indoxyl sulphate by rat renal cortical slices in vitro was also investigated. Endogenous indoxyl sulphate was found to be mainly distributed in the kidney. The rate of elimination of indoxyl sulphate from plasma was lower in CRF rats compared with sham-operated rats. The majority of intact indoxyl sulphate was excreted in the urine. In renal cortical slice experiments, uptake of indoxyl sulphate was a saturable process with a K(m) of 43.0 microm. Furthermore, sulphate conjugates, such as oestrone sulphate and dehydroepiandrosterone sulphate, inhibited the uptake of indoxyl sulphate to a greater extent than PAH. Thus, indoxyl sulphate is primarily eliminated from the plasma via the kidney by active tubular secretion, and renal uptake of indoxyl sulphate appears to be mediated by an organic anion transport system with a high affinity for oestrone sulphate and dehydroepiandrosterone sulphate.     

Proliferation in the posterior region of the lens of c-maf-/- mice

PURPOSE: To examine the involvement of the c-maf gene in the proliferation of the lens cells. METHODS: Eyes of the E13 and E18 stages of the wild-type and c-maf-/- mice were analyzed by BrdU incorporation assay, TUNEL assay and immunocytochemistry using a anti-P27(KIP1) and a anti-P57(KIP2) antibody. RESULTS: In the E13 and E18 c-maf mutant lens, BrdU-positive cells were detected at the posterior region of the lens. Cell-cycle inhibitor P27(KIP1) and P57(KIP2) were expressed in the equatorial and posterior region of the lens of both wild-type and c-maf-/- lenses. CONCLUSION: These results suggest that the expression of c-maf is required for differentiation and cell cycle arrest of lens fiber cells. It is also suggested that P27(KIP1) and P57(KIP2) were not involved in the continued proliferation of posterior region of the c-maf-/- lens.     

Expression of corticotropin releasing factor (CRF), urocortin and CRF type 1 receptors in hypothalamic-hypophyseal systems under osmotic stimulation

The expression of corticotropin releasing factor (CRF) and urocortin in hypothalamic magnocellular neurones increases in response to osmotic challenge. To gain a better understanding of the physiological roles of CRF and urocortin in fluid homeostasis, CRF, urocortin and CRF type 1 receptor (CRFR-1) gene expression was examined in the hypothalamic-hypophyseal system usingin situ and double-label in situ hybridization following chronic salt loading. CRFR-1 expression was further examined by immunohistochemistry and receptor binding. Ingestion of hypertonic saline by Sprague-Dawley rats for 7 days induced CRF mRNA exclusively in the oxytocin neurones of the magnocellular paraventricular nucleus (PVN) and the supraoptic nucleus (SON), but induced CRFR-1 mRNA in both oxytocin and vasopressin-containing magnocellular neurones. Hypertonic saline treatment also increased urocortin mRNA expression in the PVN and the SON. In the SON, urocortin was localized to vasopressin and oxytocin neurones but was rarely seen in CRF-positive cells. Changes in CRFR-1 mRNA expression in magnocellular neurones by hypertonic saline treatment were accompanied by changes in CRFR-1 protein levels and receptor binding. Hypertonic saline treatment increased CRFR-1-like immunoreactivity in the magnocellular PVN and SON, and decreased it in the parvocellular PVN. CRF receptor binding in the PVN and SON was also increased in response to osmotic stimulation. Finally, hypertonic saline treatment increased CRFR-1 mRNA, CRFR-1-like immunoreactivity and CRF receptor binding in the intermediate pituitary. These results demonstrate that the increase in the expression of CRF and urocortin message in magnocellular neurones induced by salt loading is accompanied by an increase in CRF receptor levels and binding in the hypothalamus and intermediate pituitary. Thus, CRF and urocortin may exert modulatory effects locally within magnocellular neurones as well as at the pituitary gland in response to osmotic stimulation.     

Epidemiological study on association of periodontal disease and total and differential leukocyte counts

Periodontal disease is defined as inflammation that is caused by bacteria in dental plaque. This disease is liable to be a factor contributing to the high leukocyte count over an extended period. Furthermore, a number of prospective epidemiologic studies have shown that the leukocyte count is a good predictor of ischemic heart disease (IHD). However there have been few epidemiological studies of the relationship between periodontal disease and the leukocyte count. The purpose of this study was to investigate the relationship between the total and differential leukocyte counts and oral conditions of Japanese factory workers who were classified according to their smoking habits. The 1,167 subjects were male factory workers employed with a chemical factory in Osaka, Japan. The oral conditions recorded were periodontal status (Community Periodontal Index of Treatment Needs, CPITN). The relationship between the total and differential leukocyte counts and the CPITN score of subjects who were classified according to their smoking habits was investigated in 1996 and 1997. In both the current smokers and nonsmokers, the subjects with severe periodontal disease, in contrast to the normal subjects, exhibited total leukocyte, neutrophil and monocyte counts that were significantly high for 1 year during follow-up studies. The periodontal disease is shown that causes the total leukocyte and neutrophil counts related to the development of IHD to remain at a high level.     

Chronopharmacology of analgesic effect and its tolerance induced by morphine in mice

The influence of morphine dosing time on analgesic effect after acute or chronic treatment, recovery of analgesic effect after once developed tolerance, and their pharmacological mechanisms were investigated in ICR male mice under a 12-h light/dark cycle (light on from 7:00 AM to 7:00 PM). There was a significant 24-h rhythm in the latency of thermal response at 30 min after morphine injection. The analgesic effect was significantly greater at the dark phase than at the light phase. The rhythmic pattern resembled overall the rhythm occurring in the latency of thermal response under non-drugged state. The absolute value of morphine analgesic effect (the real time spent on the hot-plate) on days 1 and 2 after morphine daily injection was significantly larger after morphine injection at 9:00 PM than after saline injection at 9:00 PM or after morphine injection at 9:00 AM. The recovery from tolerance of analgesic effect was significantly faster at the dark phase than at the light phase. The time-dependent difference in the analgesic effect after chronic treatment or recovery from tolerance is closely related to that in the expression of mu-opioid receptor. The present study suggests that 24-h rhythm of morphine analgesic effect is consistent with 24-h rhythm of mu-opioid receptor expression.