Time Sequence Studies Research Articles

Time-dependent variation of pathways and networks in a 24-hour window after cerebral ischemia-reperfusion injury.

BackgroundCerebral ischemia-reperfusion injury may simultaneously result in functional variation of multiple genes/pathways. However, most prior time-sequence studies on its pathomechanism only focused on a single gene or pathway. Our study aimed to systematically analyze the time-dependent variation in the expression of multiple pathways and networks within 24 h after cerebral ischemia-reperfusion injury.ResultsBy uploading 374 ischemia-related genes into the MetaCore software, the variation in the expression of multiple pathways and networks in 3 h, 12 h, and 24 h after cerebral ischemia-reperfusion injury had been analyzed. The conserved TNFR1-signaling pathway, among the top 10 pathways, was consistently enriched in 3 h, 12 h, and 24 h groups. Three overlapping pathways were found between 3 h and 12 h groups; 2 between 12 h and 24 h groups; and 1 between 3 h and 24 h groups. Five, 4, and 6 non-overlapping pathways were observed in 3 h, 12 h, and 24 h groups, respectively. Apart from pathways reported by earlier studies, we identified a novel pathway related to the time-dependent development of cerebral ischemia pathogenesis. The process of apoptosis stimulation by external signals, among the top 10 processes, was consistently enriched in 3 h, 12 h, and 24 h groups; 2, 1, and 2 processes overlapped between 3 h and 12 h groups, 12 h and 24 h groups, and 3 h and 24 h groups, respectively. Four, 5, and 5 non-overlapping processes were found in 3 h, 12 h and 24 h groups, respectively. The presence of apoptotic processes was observed in all the 3 groups; while anti-apoptotic processes only existed in 3 h and 12 h groups. Additionally, according to node degree, network comparison identified 1, 8,and 5 important genes or proteins (e.g. Pyk2, PKC, E2F1, and VEGF-A) in 3 h, 12 h, and 24 h groups, respectively. The Jaccard similarity index revealed a higher level of similarity between 12 h and 24 h groups than that between 3 h and 12 h groups.ConclusionTime-dependent treatment can be utilized to reduce apoptosis, which may activate anti-apoptotic pathways within 12 h after cerebral ischemia-reperfusion injury. Pathway and network analyses may help identify novel pathways and genes implicated in disease pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-015-0152-4) contains supplementary material, which is available to authorized users.

Optimization of release pattern of FGF-2 and BMP-2 for osteogenic differentiation of low-population density hMSCs

In the modern design, most delivery systems for bone regeneration focus on a single growth factor (GF) or a simple mixture of multiple GFs, overlooking the coordination of proliferation and osteogenesis induced by various factors. In this study, core-shell microspheres with poly-l-lactide core-poly(lactic-co-glycolic acid) shell were fabricated, and two GFs, basic fibroblast growth factor 2 (FGF-2) and bone morphogenetic protein 2 (BMP-2) were encapsulated into the core or/and shell. The effects of different release patterns (parallel or sequential manners) of FGF-2 and BMP-2 from these core-shell microspheres on the osteogenic differentiation of low-population density human mesenchymal stem cells (hMSCs) were investigated and the temporal organization of GF release was optimized. In vitro experiments suggested that induction of osteogenic differentiation of low-population density hMSCs by the sequential delivery of FGF-2 followed by BMP-2 from the core-shell microspheres (group S2) was much more efficient than that by the parallel release of the two factors from uniform microspheres (group U). The osteogenic induction by the sequential delivery of BMP-2 followed by FGF-2 from core-shell microspheres (group S1) was even worse than that from microspheres loaded with BMP-2 in both core and shell (group B), although comparable to the cases of parallel delivery of dual GFs (group P). This study showed the advantages of group S2 microspheres in inducing osteogenic differentiation of low-population density hMSCs and the necessity of time sequence studies in tissue engineering while multiple GFs are involved.

Electron Microscope Radiographic Studies of Lipid Synthesis, Transport, Storage, and Secretion.

Movements of labeled liver lipids were followed with time sequence studies in rats fasted and treated with ethanol.

Formation of 5α-reduced androgens in the testes and urogenital tract of the grey short-tailed opossum, Monodelphis domestica

Testicular 5alpha-reduced androgens, largely 5alpha-androstane-3alpha,17beta-diol (androstanediol), are responsible for virilisation of pouch young in one marsupial (the tammar wallaby), but are not formed until later in development in another marsupial (the brushtail possum) and in rodents. Because the mechanism of virilisation of the urogenital tract in the grey short-tailed opossum Monodelphis domestica has never been defined, androgen formation and metabolism were investigated in this species. Testis fragments from grey short-tailed opossums of a wide range of ages were incubated with [3H]-progesterone and the metabolites were separated by high-performance liquid chromatography (HPLC). The only 19-carbon metabolites identified in the youngest ages (5-26 days) and the major metabolites in adult testes were testosterone and androstenedione. At 30, 42 and 49 days of age, dihydrotestosterone and small amounts of androstanediol were present. Time-sequence studies indicated that dihydrotestosterone and androstanediol were formed from the 5alpha-reduction (and 3-keto reduction) of testosterone. In a second series of experiments, tissue fragments of a variety of urogenital tract tissues were incubated with [3H]-testosterone and the metabolites separated by HPLC. During the interval in which male urogenital tract differentiation takes place in this species (between Days 15 and 28), the major metabolite identified was dihydrotestosterone. We conclude that the timing of 5alpha-reductase expression in the testes of the grey short-tailed possum resembles that of rodents and the brushtail possum rather than that of the tammar wallaby and that dihydrotestosterone is probably the intracellular androgen responsible for virilisation of the urogenital tract in this species.

Experience with patterns of change in soil carbon resulting from forest plantation establishment in eastern Australia

Forest plantations are extensively established in eastern Australia for production of timber products and more recently, potentially for sequestration of carbon. Intensive management of these plantations involves clearing of existing vegetation, often using fire, ripping and/or mounding of the soil and weed control, followed by planting, use of fertilizers, and subsequent tending. The plantations are managed on a 10–30-year rotation and often have high growth rates and accumulation of carbon. However, after establishment, there are reduced inputs of carbon into the soil from prior vegetation or rapidly growing weeds, together with accelerated decomposition of soil organic matter as a result of disturbance, and this leads to a net loss of soil organic carbon. In some systems this loss of soil organic carbon is not balanced by carbon biomass sequestration until 5–10 years after establishment and on some sites, a reduction in soil organic carbon may remain until the end of the rotation. The patterns of accumulation and loss of carbon vary according to location, soil type and plantation management system. The effects of intensive forest establishment on soil organic carbon were evaluated in a number of studies in eastern Australia using time sequence and chronosequence studies and comparisons of plantation soils with those from adjacent undisturbed sites. There was a general pattern of reduced carbon in surface soil immediately after plantation establishment and with time this extended deeper into the soil profile. The actual quantities varied greatly depending on the soil type. The decline was primarily a result of losses of labile carbon and was greater when the previous land use had essentially been native vegetation or highly improved pastures as opposed to regrowth woodland, or native pasture, or degraded land. In the absence of further disturbance, soil organic carbon can accumulate to pre-establishment levels but many short rotation plantations are terminated prior to this being attained. The potential upper level of accumulation of soil carbon can be increased by alteration of the soil nutritional status using fertilizer application.

Evidence for heterotrimeric GTP-binding proteins in Toxoplasma gondii.

Toxoplasma gondii, an intracellular protozoan parasite, resides within a host-derived vacuole that is rapidly modified by a parasite-secreted membranous tubular network. In this study we investigated the involvement of heterotrimeric G proteins in the secretory pathway of T. gondii. Aluminum fluoride (AlFn), a specific activator of heterotrimeric G proteins, induced secretion from isolated tachyzoites of T. gondii in vitro, as seen by light optics and electron microscopy. In Western blot analyses, antibodies to G protein alpha subunits reacted with 39-42 kDa proteins from T. gondii isolates. Antibodies to G(o) alpha and Gs alpha coupled to the fluorescent probe fluorescein isothiocyanate localized to the paranuclear region of T. gondii. Gi3 alpha immunoprobes were confined to the cytoplasmic matrix of T. gondii and also labeled the parasitophorous vesicle. Fluorescein isothiocyanate-conjugated GA/1, an antipeptide antisera directed toward the GTP binding site common to G protein alpha subunits, was confined to the lateral cytoplasmic domain of the parasites where secretion is most prominent. In time-sequence studies using the GA/1 probe, the immunoreactive material shifted position during invasion of target cell to areas of active secretion.

Histology of the Pathogenesis ofMycosphaerella graminicolain Wheat

Cellular aspects of the pathogenesis of Mycosphaerella graminicola in a susceptible and resistant wheat cultivar were studied by light microscopy and scanning and transmission electron microscopy. Experiments were designed as time-sequence studies in two replications with sampling dates at 12-, 24-, and 48-h postinoculation (hpi), and 4-, 8-, 10-, 12-, 14-, and 16-days postinoculation (dpi). A separate experiment was performed to quantify the mycelial biomass in cultivars Shafir and Kavkaz/K4500 1.6.a.4 at the aforementioned intervals using a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for M. graminicola. The germination frequency of M. graminicola conidia was high in both compatible and incompatible interactions, but the infection frequency was low. Infection was strictly stomatal, but appeared to be a random process since many germ tubes crossed stomata without penetrating them. Some germ tubes formed branched structures close to or on top of stomata. These structures were small compared with the size of stomata, were formed irregularly, and were not significantly correlated with successful penetrations of the host. Multiple penetrations of stomata occurred regularly. Hyphae of M. graminicola were already observed in the substomatal cavities at 12 hpi and, at 48 hpi, hyphae had reached the nearest mesophyll cells. In the compatible response, colonization was fairly limited until 8 dpi. Hyphae grew intercellularly and in close contact with the mesophyll cells. During the 10- to 12-dpi interval, extensive host cell death occurred, which induced further colonization and, eventually, pycnidium formation in substomatal cavities. Initial and further colonization had marked effects on the number and size of the chloroplasts in the compatible interaction. Nevertheless, leaves remained green until approximately 10 dpi. The resistance response was primarily characterized by very limited colonization, mostly in the vicinity of the substomatal cavity. Quantification of the mycelial mass with ELISA revealed similar mycelial quantities in cultivars Shafir and Kavkaz/K4500 1.6.a.4 until 8 dpi. After 8 days, the mycelial quantity developed exponentially in 'Shafir,' but did not significantly increase in 'Kavkaz/K4500 1.6.a.4.'

Quercetin Down-Regulates Signal Transduction in Human Breast Carcinoma Cells

Signal transduction activity was markedly elevated in cancer cells as shown by the increased activity of enzymes utilizing 1-phosphatidylinositol, PI (PI 4-kinase and PI-4-phosphate 5-kinase) for the production of the second messenger inositol 1,4,5-trisphosphate, IP 3, in rat hepatomas (Cancer Res. 54:2611; 5574, 1994) and in human ovarian and breast carcinoma cells (Life Sci. 55:1487, 1994). Quercetin, a flavonoid, in human breast carcinoma MDA-MB-435 cells produced growth inhibition (IC 50 = 55 μM) and cytotoxicity (LC 50 = 26 μM). Quercetin inhibited PI kinase activity in extracts of breast carcinoma cells (IC 50 = 6 μM) and in cultured cells (IC 50 = 10 μM) with a minor inhibition of PIP kinase activity. IP 3 concentration decreased in parallel with PI kinase activity. In time sequence studies quercetin in breast carcinoma cells brought down PI kinase and IP 3 concentration in 60 min to 5 and 6%, respectively; PIP kinase activity was at 63% of controls. The results demonstrate for the first time in proliferating human breast carcinoma cells a reduction by quercetin of the increased capacity for signal transduction, thus providing a novel and sensitive target in cancer cells.

A method for total phosphorus determinations in nitric‐perchloric digests of pine needles and roots

Abstract A 2:1 nitric/perchloric acid plant digest was developed and tested as a replacement of the standard Kjeldahl digest for determining phosphorus concentrations in Pinus ponderosa Laws. needles and roots. Time sequence studies using the National Institute of Standards and Technology (NIST) pine standard reference material (SRM #1575) showed increased recovery of phosphorus as digest time increased from 1.5 to 16 hours. Digest intervals greater than 16 hours did not increase phosphorus recovery; the phosphorus concentration leveled off within the NIST reference material certified range. Using P ponderosa needle and root samples, a linear regression comparison determined that two sets of nitric/perchloric digests yielded 3.4% higher phosphorus concentrations than the Kjeldahl digest. However, 99 sets of nitric/perchloric digests, each containing two NIST standard reference materials (SRM), yielded an approximately normal distribution of phosphorus concentrations, all agreeing with the Kjeldahl establi...

In Vivo Effects of Recombinant Interleukin-11 on Myelopoiesis in Mice

Purified recombinant human interleukin-11 (rhuIL-11) was assessed for its in vivo effects on the proliferation and differentiation of hematopoietic progenitors as well as its capacity to accelerate the recovery of a drug-suppressed hematopoietic system. Dosage and time sequence studies demonstrated that administration of IL-11 to normal mice resulted in increases in absolute numbers of femoral marrow and splenic myeloid (granulocyte-macrophage colony-forming unit [CFU-GM], burst-forming unit-erythroid [BFU-E], CFU-granulocyte, erythroid, macrophage, megakaryocyte) progenitor cells and in stimulation of these progenitors to a higher cell cycling rate. This was associated with increased numbers of circulating neutrophils. Administration of IL-11 to mice pretreated with cyclophosphamide decreased the time required to regain normal levels of neutrophil and platelet counts in peripheral blood. In addition, IL-11 accelerated reconstitution to normal range of myeloid progenitors from bone marrow and spleen of myelosuppressed mice. These data suggest that IL-11 may play an important role in the regulation of hematopoiesis, and the application of this novel cytokine may have clinical therapeutic benefits.

In vivo effects of recombinant interleukin-11 on myelopoiesis in mice

Purified recombinant human interleukin-11 (rhuIL-11) was assessed for its in vivo effects on the proliferation and differentiation of hematopoietic progenitors as well as its capacity to accelerate the recovery of a drug-suppressed hematopoietic system. Dosage and time sequence studies demonstrated that administration of IL-11 to normal mice resulted in increases in absolute numbers of femoral marrow and splenic myeloid (granulocyte-macrophage colony-forming unit [CFU-GM], burst-forming unit-erythroid [BFU-E], CFU-granulocyte, erythroid, macrophage, megakaryocyte) progenitor cells and in stimulation of these progenitors to a higher cell cycling rate. This was associated with increased numbers of circulating neutrophils. Administration of IL-11 to mice pretreated with cyclophosphamide decreased the time required to regain normal levels of neutrophil and platelet counts in peripheral blood. In addition, IL-11 accelerated reconstitution to normal range of myeloid progenitors from bone marrow and spleen of myelosuppressed mice. These data suggest that IL-11 may play an important role in the regulation of hematopoiesis, and the application of this novel cytokine may have clinical therapeutic benefits.

Morphological aspects of the interaction of Bacillus Calmette-Guérin with urothelial bladder cells in vivo and in vitro: relevance for antitumor activity?

Intravesical administration of Bacillus Calmette-Guérin (BCG) has been shown to be effective in the treatment of patients with superficial bladder cancer. For a better understanding of the mechanism of this antitumor activity, scanning and transmission electron microscope (SEM, TEM) studies were carried out to investigate morphological aspects of the interaction of BCG with the bladder wall in vivo and in vitro. Adherence of BCG to the bladder wall in vivo was studied 1 and 24 h after single or multiple (6x) BCG instillations in intact and in electrocauterized guinea pig bladders. Despite extensive search with SEM for its presence, virtually no BCG was found on the intact urothelium, and BCG was only occasionally observed in the coagulation lesions. SEM and TEM studies revealed adherence and phagocytosis of BCG by the T24 human bladder carcinoma cell line in vitro. Time sequence studies on the phagocytosis and fate of BCG showed that T24 cells are capable of progressively degrading the mycobacteria in phagolysosomes. However, BCG did not alter MHC class II antigen expression on T24 cells in vitro. In contrast, 54 urine sediments and bladder washings of 11 bladder cancer patients, taken prior to or after several intravesical BCG instillations, failed to demonstrate urothelial (tumor) cells showing evidence of BCG phagocytosis (682 cells screened by TEM), while BCG was phagocytized avidly by leukocytes. These data suggest that a direct interaction of BCG with urothelial bladder cells in vivo can be called in question.

Temporal Effect of Prolactin on the Activities of Lactose Synthetase, -Lactalbumin, and Galactosyl Transferase in Mouse Mammary Gland Explants

The onset of the prolactin (PRL) stimulation of lactose synthesis is between 4 and 8 hr after adding PRL to cultured mouse mammary tissues. The synthesis of lactose is catalyzed by the enzyme lactose synthetase, which is composed of two parts, alpha-lactalbumin and galactosyl transferase. In time-sequence studies, it was found that the activity of galactosyl transferase is enhanced by PRL in concert with the onset of the PRL stimulation of lactose synthesis. In contrast, the earliest detectable effect of PRL on alpha-lactalbumin activity occurred 24 hr after adding PRL to the cultures. It is, therefore, apparent that the rate-limiting component for the PRL stimulation of lactose synthesis in cultured mouse mammary tissues is galactosyl transferase activity.

Cytometry and Time-Dependent Variations in Peripheral Blood and Bone Marrow Cells: A Literature Review and Relevance to the Chronotherapy of Cancer

By flow cytometry of individual cells, multiple cell properties can be analyzed. Such parameters may be important in relation to cytotoxic treatment of cancer. For example, DNA measurements will answer questions regarding cell kinetics. Myelosuppression is the major dose-limiting toxicity during cancer treatment. Therefore, the study of cell cycle parameters in bone marrow cells is highly relevant. However, inattention to the existence and potential importance of biological rhythms may introduce artifacts and misleading results. The literature of rhythms in hematology is reviewed. Time-dependent variations in hematological variables have been extensively studied and rhythms have been described for all kinds of blood cells. Also the numbers of hemopoietic stem cells in the bone marrow undergo circadian variations. Our group has shown how such variations change with aging in mice. The relevance of time sequence studies in aging research of hemopoiesis was clearly demonstrated. In animal studies using cytometry, our group has demonstrated extensive circadian variations in cell cycle distribution of bone marrow cells, especially the DNA synthesis (S-phase). In humans a few and rather small time sequence studies of the bone marrow have been performed, so far. In this overview the clinical implications of circadian rhythms of S-phase variations measured by flow cytometry of human bone marrow cells are discussed. Male volunteers were examined every 4 h around-the-clock. The data indicated a lower proliferative activity during night, suggesting the possibility of reducing the bone marrow toxicity to cancer treatment when taking these time-dependent variations into consideration.

Generation of chemiluminescence in experimental autoimmune uveitis.

Using experimental model of uveitis, the inflammation-associated chemiluminescence was measured by luminol amplification method. The induction of uveitis in Lewis rats was carried out by immunizing bovine S-antigen in complete Freund's adjuvant on hind foot-pad. The chemiluminescence activity from six sets of retina and choroid measured at the peak of inflammation was found to be in the range of 300,000-400,000 counts, amounting to nearly 20-fold increase from non-immunizated control animals under the same conditions. The phagocyte-derived superoxide anion and hydroxyl radical appear to be the two major radicals in the chemiluminescent species. This was revealed by in vitro suppression of chemiluminescence by 5,400 units of superoxide dismutase and 10 mM of D-mannitol. The decreases in counts were 21% and 35% for superoxide dismutase and D-mannitol respectively. The presence of hydrogen peroxide was not established since catalase with dose up to 20,000 units did not cause any significant suppression in counts. In the time sequence studies covering the entire course of uveitis development, the level of chemiluminescent species at day 9 postimmunization increased to 1.5-fold that of day 0, continued to increase to a maximum of 18-fold on day 12 and decreased slowly to about 3-fold in day 19. The pattern of increase appears to coincide with our previous findings in the number of polymorphonuclear leukocytes, the extent of membrane lipid peroxidation and the degree of retinal degeneration. Thus, these radicals play an important role in initiation as well as perpetuation of the membrane oxidative processes that lead to retinal degeneration.

Studies on the mechanism by which prolactin regulates protein, RNA, and DNA synthesis in Nb2 node lymphoma cells

Specific aspects of the prolactin stimulation of RNA, DNA and protein synthesis in the Nb2 node lymphoma cell line were determined. In time sequence studies the onset of the prolactin stimulation of the incorporation of radiolabeled precursors into these macromolecules was found to be 0.5-1 h for [3H]uridine incorporation into RNA, 1-2 h for [3H]leucine incorporation into protein, and 4-8 h for [3H]thymidine incorporation into DNA. The total DNA content of the cell cultures was increased by 12-18 hours after addition of prolactin. Amiloride, an inhibitor of the plasma-membrane-bound Na+/H+ antiporter, was found to inhibit the mitogenic effects of prolactin. Amiloride was also found to inhibit the prolactin stimulation of DNA, RNA and protein synthesis, thus suggesting that the initial regulation of the Na+/H+ antiporter may initiate these responses as well as the mitogenic effect of prolactin. In contrast, H-7, a drug which inhibits protein kinase C, had no effect on the magnitude of the prolactin stimulation of DNA, RNA or protein synthesis at a drug concentration (100 muM) that abolished the mitogenic effect of prolactin. The early effects of prolactin on RNA, DNA and protein synthesis would therefore appear not to involve an activation of protein kinase C.

Rhythmic variations of different hemopoietic cell lines and maturation stages in aging mice

Non-proliferative and proliferative myeloid, and lymphoid and erythroid bone marrow cells were studied in aging female C3H mice. A chronobiological approach was used and mice aged 16, 21 and 26 months were examined vs. 3 month-old mice every 3 h during the 24-h period in three different experiments. Significant circadian fluctuations were observed in most of the cell populations, even in the oldest mice. The rhythmicity patterns might be different at different times of the year, and in young mice seasonal fluctuations in the 24-h mean values were observed. The absolute numbers of the 24-h means seemed to be highest at 21 months of age in all cell lines and maturation stages. Sinus function fitting indicated a decline of the amplitudes in aging mice. Minor age-related phase-differences were indicated in some populations. However, the fitting of original data to single sinus functions was variable and often obscured important features in the cell number variations. The present investigation illustrates the importance of performing time-sequence studies in hematology.

Experimental indications of a low undercooling instability in the cellular mechanism of discontinuous precipitation

For the formation of well formed lamellar cells during discontinuous precipitation, there must be a “cooperation” between the migration of the grain boundary and the morphological development of the precipitating phase. If the grain boundary velocity is greater than the velocity of the precipitate growth projected along the direction of grain boundary motion, large curvatures may develop leading to temporal oscillations in grain boundary position and/or the eventual separation of the grain boundary from many of the precipitates. For the larger undercoolings, Onsorio (10) has shown by time sequence studies that this does not occur. However, for smaller undercoolings the grain boundary is migrating faster than the projected velocity of the precipitate growth interface, the curvature of the grain boundary increases until the grain boundary separates from the precipitate decreasing the driving force for migration. The cooperation between grain boundary migration and precipitation will break down and the cellular mechanism will be “unstable”. We have probed the physical basis of the cellular transformation by examining changes in microstructural development as the undercooling is decreased. The expectation that the cooperative effects would break down as the system approached equilibrium (decreasing undercooling) was fulfilled. This leads us to conclude that the cellular mechanism belongs to a general class of cooperative phenomena which are observed to occur in nature and may be successfully investigated using the same general approach outlined in (3) and (4). We do not wish to convey the impression that because the cellular transformation has been grouped (by the authors) with a class of phenomena generally referred to as “dissipative” (11) that an entropy flux criteria is all that is needed to understand the process. On the contrary, the experimental results presented here should be thought of as guidelines to the development of a microscopic kinetic theory which predicts the onset of the cellular mechanism as the undercooling is increased. As suggested by the experiments, this would require (1) explaining why the grain boundary migrates when essentially equiaxed precipitation occurs, and (2) why the precipitation morphology becomes elongated as the undercooling is increased. In other terms, to understand the cellular transformation we need to understand the transitions from the mechanisms 1–3 as the undercooling is increased. Any application of a macroscopic non-equilibrium stability criteria should only be attempted after the kinetic theory has been sufficiently developed.

Tiazofurin-induced selective depression of NAD content in hepatoma 3924A

The NAD content in hepatoma 3924A was approximately 40% of that in the liver of ACI N rats bearing this hepatoma. Treatment of tumor-bearing rats with tiazofurin decreased NAD pools in the hepatoma, but no change was apparent in the liver. In a dose-response study, injection of varying amounts of the drug decreased NAD pools in the hepatoma in a dose-dependent fashion. In time-sequence studies, a single drug dose (200 mg/kg) depressed NAD pools in the hepatoma from 2 to 24 h after injection to approximately 50% of control at the lowest point before returning to control range at 48 h. The tiazofurin-induced depletion of NAD pools in the hepatoma to approximately 20% of that of normal liver might play a role in the anti-cancer action and toxicity of this drug.

Moistness, more than temperature variation, prevents autolysis of pulp tissue in avulsed teeth

Through the years, evaluators of pulp tissue have been plagued distinguishing between true pathology and artifacts of fixation, processing, and sectioning. Studies have been published stressing techniques for immediate fixation of pulps after extraction or resection of jaw blocks, even following perfusion, to obtain the maximum degree of fixation. To facilitate the penetration of the fixative it has been recommended to cut off the root end immediately; the longer the root and the older the tooth the more of the root end is cut off; cut at high speed with an air-water spray deep class V cavity preparations; or slice the proximal surfaces (1-3) to open up dentinal tubules. Time sequence studies have shown increasing degrees of autolysis that can develop in pulp tissue when an extracted tooth is permitted to bench-dry at room temperature and then cracked open and placed in formalin for immediate fixation (1,2). Such intensional studies help the microscopist recognize such characteristics due unintentionally to delayed or inadequate fixation occurring in surgical specimens submitted for histopathological evaluation. When such pulps are examined, one does not know how quickly the fixative penetrated the tooth and reached the pulp. Rapidity of fixative penetration is the important factor. In the Pulp Registry we have been impressed with the preservation of pulps extirpated from avulsed teeth that have been replanted quickly and treated endodontically several days later. Appreciating the fact that there has been no blood supply to the replanted avulsed tooth, how does one explain the lack of the usual autolytic changes so frequently described. In order to preserve the surviving attached periodontal tissues which enhance replantation success and reduces the potential for resorption, it is now recommended that the avulsed tooth be placed back in the socket or at least held in the oral vestibule, or wrapped in gauze and placed in saline or milk (4, 5) until the tooth is replanted. Possibly the pulp tissues within avulsed teeth treated as described above also benefit in terms of tissue preservation. By permitting an extracted tooth to bench-dry for periods of 45 min to 7 h, before splitting the tooth to obtain immediate pulp fixation, varying degrees of 36o reticulation of the individual pulp cells and separation of the odontoblastic layer from the predentin occurs until the classical picture of reticular atrophy develops (1, 2). The purpose of this study was to establish whether body temperature could be one of the factors that protects pulp tissues from autolytic changes as compared with room temperature.