VP-16-induced nucleotide pool changes and poly(ADP-ribose) synthesis: the role of VP-16 in interphase death

Exposure of human promyelocytic leukemia cell line (HL-60) to VP-16 resulted in accumulation of DNA strand breaks. Concomitantly, intracellular NAD levels fell at 1 h, followed by declines in ATP at 2 h and in GTP, CTP, and UTP at 3 h. Furthermore, marked morphological changes, such as loss of microvilli or bleb formation, appeared at 4 h and cell death by 8-10 h. The addition of an inhibitor of poly(ADP-ribose) polymerase, 3-aminobenzamide (5 mM), theophylline (2 mM), or thymidine (1 mM), prevented these sequential reductions of nucleotide pools and cell death. In fact, the activation of poly(ADP-ribose) synthesis was detectable within a few hours after treatment with VP-16, although it was smaller than that induced by N-methyl-N'-nitro-N-nitrosoguanidine. These results may suggest the possible role of activation of poly(ADP-ribosyl)ation in VP-16-induced nucleotide pool changes and subsequent interphase death.     

An improved technique for dissociating hepatocytes from fixed mouse liver for cytochemistry

A refined version of a method described previously for dissociating hepatocytes from fixed liver is described. The objective was to develop a procedure that would dispense with the postosmication of previously fixed tissue. In the new procedure fixed liver blocks are wrapped with a quadruple layer of nylon cloth, and, by squeezing them in a buffer solution, individually dissociated cells pass through the cloth without significant damage. The procedure makes it possible to dissociate liver tissue fixed with modified Karnovsky's fixative, Zamboni's fixative or cacodylate buffered glutaraldehyde. The subsequent compatibility of the single cells obtained with many cytochemical procedures has been confirmed.     

Immunohistochemical Detection of DNA Replication in Mouse Uterine Cells by Bromodeoxyuridine Labeling of Wax- and Resin-Embedded Tissue Sections

TO apply the bromodeoxyuridine (BrdU) labeling method using a monoclonal antibody to the study of cell proliferation in the mouse uterus, methods of fixation and embedding of tissues and of immunofluorescent staining were compared in terms of the rate of detection of labeled cells and specificity and stability of fluorescence obtained. BrdU was administered intravenously 2 hr before death and uterine blocks were embedded in polyester wax and Technovit resin after fixation in formalin and periodate-lysine-paraformaldehyde, respectively. The indirect method with anti-BrdU and fluorescein isothiocyanate (FITC) conjugated antimouse IgG antisera and the direct method with FITC conjugated anti-BrdU antibody were applied to both wax- and resin-embedded sections. Labeled and total cells were counted in luminal and glandular epithelia and stromata adjoining them. Counterstaining with hematoxylin for counting total cells produced intense fluorescence over the whole of resin sections and made counting of labeled cells impossible. On wax sections, on the other hand, the results were satisfactory, although the number of labeled cells detected was decreased slightly. In wax sections fluorescence due to nuclear incorporation of BrdU in the indirect method could be easily distinguished from the cytoplasmic or extracellular emission seen in some cells by its location and characteristic color. In resin sections, however, more careful observation was needed since the second antibody used in the indirect method cross-reacted with IgG in eosinophils and produced cyctoplasmic fluorescence of the same color. By the indirect method greater numbers of labeled cells were detected in wax sections than in resin sections. The difference was distinct in tissues with extensive cell proliferation. By the direct method the fluorescence obtained was weaker and apt to fade more quickly than that obtained by the indirect method; use of the direct method reduced the number of labeled cells detected in both wax- and resin-embedded sections.     

Metabolism of urea in Chlorella ellipsoidea

The effect of cyanide on ammonia and urea metabolism was studiedwith intact cells of Chlorella ellipsoidea Gerneck, a greenalga which apparently lacks urease. Ammonia uptake was inhibited more readily by cyanide than wasurea uptake. Urea uptake was stimulated by lower concentrationsof cyanide. The addition of cyanide caused the formation ofammonia from some cellular nitrogenous compounds. In the presenceof exogenously added urea, the molar ratio of ammonia accumulatedin the medium to urea taken up exceeded 2.0 as the cyanide concentrationincreased. However, the molar ratio of ammonia actually producedfrom urea nitrogen to urea taken up was less than 1.35 at anyconcentration of cyanide tested. In the presence of higher concentrationsof cyanide, the rate of incorporation of ¹⁵N into amino acidsfrom ¹⁵N-urea was higher than that from ¹⁵N-ammonium sulfate. The results suggest that Chlorella ellipsoidea possesses a pathwaythrough which urea nitrogen is assimilated directly withouta preliminary breakdown to ammonia. (Received October 18, 1976; )     

Skewed X-chromosome inactivation causes intra-familial phenotypic variation of an EBP mutation in a family with X-linked dominant chondrodysplasia punctata

X-linked dominant chondrodysplasia punctata (CDPX2) is a skeletal dysplasia characterized by stippled epiphyses, cataracts, alopecia and skin lesions, including ichthyosis. CDPX2 exhibits a number of perplexing clinical features, such as intra- and inter-familial variation, anticipation, incomplete penetrance and possible gonadal and somatic mosaicism. Recently, mutations in the gene encoding Delta8,Delta7 sterol isomerase/emopamil-binding protein (EBP) have been identified in CDPX2. To better understand the genetics of CDPX2, we examined the entire EBP gene by direct sequencing in four CDPX2 patients. We found EBP mutations in all four patients, including three novel mutations: IVS3+1G>A, Y165C and W82C. Surprisingly, a known mutation (R147H) was identified in a patient and her clinically unaffected mother. Expression analysis revealed the mutant allele was predominantly expressed in the patient, while both alleles were expressed in the mother. Methylation analysis revealed that the wild-type allele was predominantly inactivated in the patient, while the mutated allele was predominantly inactivated in her mother. Thus, differences in expression of the mutated allele caused by skewed X-chromosome inactivation produced the diverse phenotypes within the family. Our findings could explain some of the perplexing features of CDPX2. The possibility that an apparently normal parent is a carrier should be considered when examining seemingly sporadic cases and providing genetic counseling to CDPX2 families.     

Up-regulation of hyaluronan receptors in Toxoplasma gondii-infected monocytic cells

The apicomplexan, obligate intracellular parasite Toxoplasma gondii orally infects humans and animals. The parasites cross the intestinal epithelium, invade leukocytes in the general circulation and then disseminate into the peripheral organs. The mechanism of extravasation of the infected leukocytes, however, remains poorly understood. It is known that adhesion of leukocytes to extracellular matrix (ECM) is an important factor in extravasation, and CD44 and ICAM-1 on the leukocyte surface are known receptors for hyaluronan (HA), an ECM component. In this study, we demonstrated up-regulation of CD44 and ICAM-1 expression on the surface of T. gondii-infected human monocytic THP-1 cells and fresh isolated human monocyte. T. gondii-infected THP-1 cells adhered more efficiently to immobilized HA than did non-infected cells. T. gondii-infected monocytes in the general circulation might preferentially adhere to the ECM and migrate out from blood vessels, so transporting parasites into the peripheral organs.     

A recurrent mutation in type II collagen gene causes Legg-Calvé-Perthes disease in a Japanese family

Legg-Calvé-Perthes disease (LCPD) is a common childhood hip disorder characterized by sequential stages of involvement of the capital femoral epiphyses, including subchondral fracture, fragmentation, re-ossification and healing with residual deformity. Most cases are sporadic, but familial cases have been described, with some families having multiple affected members. Genetic factors have been implicated in the etiology of LCPD, but the causal gene has not been identified. We have located a missense mutation (p.G1170S) in the type II collagen gene (COL2A1) in a Japanese family with an autosomal dominant hip disorder manifesting as LCPD and showing considerable intra-familial phenotypic variation. This is the first report of a mutation in hereditary LCPD. COL2A1 mutations may be more common in LCPD patients than currently thought, particularly in familial and/or bilateral cases.     

Development of a new class of benzoylpyrrole-based PPARα/γ activators

Starting with a subtle blood glucose-lowering effect of a TGF-β inhibitor, we designed and synthesized a series of benzoylpyrrole-based carboxylic acids as PPARs activators. Among these compounds, 10sNa exhibited favorable blood glucose-lowering effect without body weight gain. We assume that the beneficial effect of 10sNa is attributed to not only its compound PPARα agonistic activity but also its PPARγ partial agonistic activity.     

Dosing-time-dependent differences in lipopolysaccharide-induced liver injury in rats

Dosing-time-dependent differences in lipopolysaccharide (LPS)-induced liver injury were examined in rats housed under a 12 h light:dark (LD) cycle. LPS (5 mg/kg) was intravenously injected into different groups of rats at 2, 14, or 20 h after light on (HALO). Elevations in serum liver enzymes after 14 HALO were significantly greater than those after 2 HALO. These parameters were lower in rats given LPS at 20 HALO, compared to 14 HALO. The number of polymorphonuclear cells (PMN) in the liver and the amount of hepatic myeloperoxidase activity, which reflects the number of PMN in liver tissues, was significantly greater in the 14 than in the 2 HALO group. In addition, hepatic interleukin-6 (IL-6) production in the 14 HALO group was enhanced compared to that in the 2 HALO trial. These results suggest that LPS-induced liver injury is greater during the early active than during the early resting period. Dosing-time-dependent variation in the accumulation of PMN in the liver and, potentially, subsequent IL-6 production in liver tissues might be involved in this phenomenon.