Growth of epizootic hemorrhagic disease, akabane, and ephemeral fever viruses in Aedes albopictus cells maintained at various temperatures

The growth curves of one epizootic hemorrhagic disease (EHD) virus serotype (Reoviridae), two Akabane virus strains (Bunyaviridae) and three bovine ephemeral fever (BEF) group viruses (Rhabdoviridae) were determined in Aedes albopictus cells maintained at 15, 20, 28 and 33 degrees C. Ae albopictus cells supported the growth of all the viruses although not necessarily at all temperatures. Because none of the viruses exhibited cytopathic effect in Ae albopictus cells, growth was assayed in baby hamster kidney 21 (BHK21) cells maintained at 37 degrees C. The temperature at which the Ae albopictus cells were maintained had a marked effect on the growth and yield for each virus studied. EHD virus was heat-stable and grew after 4 days at 28 and 33 degrees C, and after 8 days at 20 degrees C. No growth was recorded up to 12 days at 15 degrees C. The two Akabane viruses were heat-sensitive and exhibited different growth patterns. One strain (B8935) showed no growth at 15 degrees C and only minimal growth at 20, 28 and 33 degrees C. The other strain (CSIRO 16) showed growth after 1-2 days at all temperatures with higher titres reached at 15 and 20 degrees C than at 28 and 33 degrees C. The BEF group viruses grew to approximately the same titres at all temperatures. At the higher temperatures (28 and 33 degrees C) most of BEF group viruses had disappeared within 9 days. In contrast at the lower temperatures (15 and 20 degrees C), there was still virus present 18 days after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of photoperiod and cold acclimation upon plasma free fatty acid levels in the white rat.

1. Animals were acclimated at 3 +/- 1 degrees C and at room temperature (22 degrees C) for 3 weeks. 2. At each acclimation temperature animals were maintained under either an 8:16 L:D cycle or a 16:8 L:D cycle. 3. Blood samples were taken before and after exposure to -38 degrees C for 30 min. 4. Free fatty acid levels were greatest in cold acclimated animals which were maintained on a short light cycle. 5. Interaction between acclimation and photoperiod was apparent.     

Low ambient temperature accelerates short-day responses in Siberian hamsters by altering responsiveness to melatonin

Exposure to low ambient temperatures (Ta) accelerates appearance of the winter phenotype in Siberian hamsters transferred from long to short day lengths. Because melatonin transduces the effects of day length on the neuroendocrine axis, the authors assessed whether low Ta promotes the transition to winterlike traits by accelerating the onset of increased nocturnal melatonin secretion or by enhancing responsiveness to melatonin in short day lengths. Male hamsters were transferred from 16L (16 h light/day) to 8L (8 h light/day) photoperiods and held at 5 degrees C or 22 degrees C. Locomotor activity was recorded continuously, and body mass, testis size, and pelage color were determined biweekly for 8 weeks. The duration of nocturnal locomotion (alpha), a reliable indicator of the duration of nocturnal melatonin secretion, lengthened significantly earlier in hamsters exposed to a Ta of 5 degrees C than 22 degrees C. Cold exposure increased the proportion of hamsters that were photoresponsive: gonadal regression in short days increased from 44% at 22 degrees C to 81% at 5 degrees C (p < 0.05); low Ta did not, however, accelerate testicular regression in animals that were photoresponsive. Nonphotoresponsive animals at 5 degrees C temporarily had longer alphas during the first 4 weeks in short days and significant decreases in body mass and testicular size that were reversed during the ensuing weeks when alpha decreased. In a 2nd experiment, pinealectomized male hamsters infused for 10 h/day with melatonin for 2 weeks had significantly lower body and testes masses when maintained at 5 degrees C but not 22 degrees C. Low-ambient temperature appears to accelerate the appearance of the winter phenotype primarily by increasing target tissue responsiveness to melatonin and to a lesser extent by augmenting the rate at which the duration of nocturnal melatonin secretion increases in short day lengths.     

Thermostability of Japanese encephalitis vaccine produced in India.

Different batches of bulk vaccine, final bulk at in-process level, finished freeze-dried and reconstituted Japanese encephalitis vaccine were assayed for their stability at temperatures of 22, 37 and 40 degrees C. After ultrazonal purification of 50 times concentrated brain suspension, JE Bulk vaccine was found to be stable for up to 2 years at 4 degrees C, however, the percentage loss in potency (log 10 N antibody titre) after 2.5 years was 24%. Three-times concentrated final bulk showed rapid deterioration by the fourth week at 37 and 40 degrees C. Freeze-dried JE vaccine maintained at 22 degrees C for 28 weeks did not show perceptible deterioration. At 37 degrees C, the same vaccine started showing deterioration (14%) after 8 weeks whereas at 40 degrees C the loss of potency was 24% after 8 weeks. The freeze-dried vaccine was found to be stable for up to 2 weeks duration at 40 degrees C.     

Introduction of wild-type p53 in a human ovarian cancer cell line not expressing endogenous p53.

Utilizing a temperature sensitive p53 mutant (pLTRp53cGval135) which expresses mutant p53 at 37 degrees C and a wild-type like p53 at 32 degrees C, we transfected a human ovarian cancer cell line (SKOV3) which does not express endogenous p53. Among the different clones obtained, we selected three clones. Two were obtained from simultaneous transfection of p53 and neomycin resistance expression plasmids (SK23a and SK9), the other was obtained from transfection experiments utilizing the neomycin resistance gene only (SKN). Introduction of mutant p53 did not alter the morphology or growth characteristics of this ovarian cancer cell line. Upon shifting to the permissive temperature, a dramatic change in morphology and growth rate was observed in SK23a and SK9 cells that is associated with the presence of a wild-type like p53. SKN and SKOV3 cells maintained at 32 degrees C did not change morphology and only slightly reduced proliferation. Both SK23a and SK9 cells did not show evidence of apoptosis when measured up to 72 hours of maintenance at 32 degrees C. In contrast to what observed in other cell lines, SK23a and SK9 cells maintained at 32 degrees C were not blocked in G1, but they were accumulated in G2-M. This accumulation was transient and could be due either to a blockade or to a delay in the G2 progression. No down-regulation of c-myc was observed in p53 expressing clones when shifted to the permissive temperature. In these conditions gadd45 mRNA expression was highly stimulated in SK9 and SK23a cells but not in SKN cells. In both clones Gas1 mRNA was not detected either at 37 degrees C or 32 degrees C. This system represents a new and useful model for studying the effect of the absence of p53 (SKOV3 or SKN), presence of mutated p53 (SK23a and SK9 kept at 37 degrees C) or wild type p53 (SK23a and SK9 kept at 32 degrees C) on the mechanism of response of cancer cells to DNA damaging agents.     

Effects of duration of daily illumination on reproductive organs and fertility of the meadow vole (Microtus pennsylvanicus).

The objective of this study was to determine the optimum simple, constant photoperiod for voles in a laboratory colony. Voles from an established colony maintained at 22 degrees C with a photoperiod of 14:10 hours of light:dark were transferred at 50 days of age to a photoperiod of 12:12, 14:10, 16:8 or 18:6 hours of light:dark. From Day 96 to Day 102, the 9--10 females per treatment group were paired with a male. Body and gonadal weights, spermatogenesis, ovarian activity and pregnancy were evaluated on Day 110. Reproductive function and body weight of both male and female voles maintained on a photoperiod of 16:8 exceeded (p less than 0.05) values for voles exposed to 12:12 hours of light:dark and tended to be more favorable than for voles in the 14:10 and 18:6 groups.     

Effect of precocene II on fatty acid metabolism in the pea aphid, Acyrthosiphon pisum, under cold stress

Pea aphids, Acyrthosiphon pisum (Harris), reared at 10 degrees C contain higher levels of fatty acids than those reared at 25 degrees C. This is primarily the result of an accumulation of triacylglycerols containing myristic acid. When aphids reared at 25 degrees C were transferred to 10 degrees C there was a gradual increase in triacylglycerol content that reached a maximum at 16 days post-transfer. Treatment of aphids with precocene II prior to transfer to 10 degrees C blocked the accumulation of fatty acids including myristic acid. A single application of 2 microg precocene II/aphid or two applications of 0.5 microg precocene II/ aphid administered on consecutive days resulted in aphids moved to 10 degrees C maintaining the same fatty acid profile as aphids maintained at 25 degrees C. Aphids that were treated with precocene II and maintained at 25 degrees C did not show changes in fatty acid profiles. Rearing aphids at 10 degrees C resulted in lower rates of reproduction and lower total numbers of progeny with longer longevity. Treatment with precocene II significantly decreased the total number of progeny produced at both temperatures. Precocene II did not reduce life span of aphids reared at 25 degrees C, however, the life span of treated aphids reared at 10 degrees C was decreased. The mechanism by which precocene II prevents the accumulation of myristic acid in aphids reared at 10 degrees C remains to be determined.     

Effect of incubation temperature on zearalenone production by strains of Fusarium crookwellense.

After 6 weeks incubation on rice 2 strains of Fusarium crookwellense produced more zearalenone (6060-5010 mg/kg dry wt of culture) at ambient temperature (16-29 degrees C) in daylight than at ambient temperature (18-23 degrees C) in darkness or at controlled temperatures of 11 degrees C, 20 degrees C or 25 degrees C in darkness. Yields at 25 degrees C were low. Incubation at 11 degrees C during the second 3 weeks incubation increased yields only when preliminary incubation had been at 25 degrees C. After 6 weeks incubation at controlled temperatures in darkness, 4 strains produced most zearalenone at 20 degrees C (2460-21 360 mg/kg), 1 strain at 11 degrees C (6570 mg/kg). Yields at a temperature oscillating daily from 10-20 degrees C were less than at 15 degrees C. One of the 5 strains produced appreciable amounts of a-zearlaenol (1645 mg/kg at 20 degrees C) and 2 of nivalenol (340 and 499 mg/kg at 20 degrees C).     

Induction and development of winter larval diapause in a drosophilid fly,Chymomyza costata

Photoperiodic response during induction of larval hibernal diapause of Chymomyza costata was characterized and the course of diapause development was analyzed in the laboratory. C. costata becomes sensitive to photoperiodic stimuli during an unspecified stage of its early development (embryo, 1st larval instar); the sensitivity gradually increases during the 2nd and early 3rd larval instars and reaches its maximum just before the moment when it abruptly ceases at the age of 15-19 days after oviposition. Diapause intensifies during a period of 2-3 weeks after induction and, later, is maintained without apparent development until death (between 150 and 250 days) under 18 degrees C and a short-day photoperiod (L10:D14, SD). Diapause may be terminated in a horotelic process by exposure to a low temperature (2 degrees C) during which larvae subsequently (1) synchronize their post-diapause development (requires up to 14 days of chilling), (2) lose photoperiodic sensitivity (2 months), and finally (3) terminate diapause (5 months). Alternatively, diapause may be terminated in a tachytelic process by exposure to a high temperature (18 degrees C) and long-day photoperiod (L16:D8, LD) during which no synchronization occurs and pupariation takes place after a mean of 25.2 days (with a broad range from 8 to more than 50 days). Larvae that are transferred from LD to SD during their sensitive period switch their developmental programming from pupariation to diapause. Proliferation of adult primordial structures (imaginal discs, neuroblasts) slows down within 1 day after transfer. In contrast, whole body growth continues for at least 3 days before its rate slows down and matches the rate characteristic for SD conditions.     

Analysis of fatty acid composition of Candida species by gas-liquid chromatography using a polar column

The fatty acid composition of representative Candida species was examined by gas-liquid chromatography (GLC) using a polar column. The major fatty acids were C14:0, C16:0, C18:0 saturated, C16:1 and C18:1 monoenoic series, with or without C18 polyunsaturated acids (C18:2 and C18:3). In Torulopsis glabrata and Saccharomyces cerevisiae the C18:2 and C18:3 acids were not found, but the C10:0 and C12:0 acids were detected in S. cerevisiae. These results indicated that the Candida genus could be distinguished from Torulopsis and Saccharomyces genera by GLC analysis of fatty acids. Quantitative differences in the fatty acid composition between cells grown at high temperature (37 degrees C) and low temperature (25 degrees C) were found generally in Candida species, and the amounts of C18 polyunsaturated acids (C18:2 and C18:3) increased in the cells grown at 25 degrees C. Each Candida species showed a characteristic profile in fatty acid composition. Determination of the cellular fatty acid composition in Candida species is likely to be useful for the grouping or chemotaxonomy of newer isolates of Candida species.     

A cell cycle ts mutant, tsJT16, is defective in p70 synthesis through protein kinase C-dependent and -independent pathways.

tsJT16 is a G0/G1 ts mutant from the Fischer rat fibroblast line. It has a ts defect in a function operating early after growth stimulation with fetal bovine serum (FBS). A primarily induced gene product, p70, was not synthesized at 40 degrees C after stimulation with serum, while c-fos and c-myc mRNAs accumulated under the same condition. This paper reports that p70 was synthesized following stimulation of G0-arrested cells with platelet-derived growth factor, epidermal growth factor (EGF), and 12-0-tetradecanoylphorbol-13-acetate (TPA) at 34 degrees C, but not at 40 degrees C. However, it was synthesized at both temperatures after addition of A23187. In protein kinase C-deprived cells, peptide growth factors and A23187 induced p70 at 34 degrees C, whereas TPA did not. Fibroblast growth factor and insulin did not induce p70. Induction of c-fos and c-myc occurred at both temperatures after the stimulation with FBS, TPA or A23187. These results indicated that the defect in tsJT16 to induce p70 is likely to be located at the common downstream of protein kinase C-dependent and -independent pathways, but is independent from the pathway of calcium mobilization.     

Effect of prior refrigeration on botulinal outgrowth in perishable canned cured meat when temperature abused.

Perishable canned cured meat inoculated with Clostridium botulinum spores was placed at 4.4 or 10 degrees C after manufacture. Spore germination occurred at 10 degrees C. The germinated cell count remained stable over a period of 16 to 18 weeks. During that time period the inhibitory system and residual nitrite descreased. These factors combine to make perishable canned cured meats more prone to spoilage and potential hazard if they are temperature abused after a period of refrigerated storage.     

Effect of prior refrigeration on botulinal outgrowth in perishable canned cured meat when temperature abused.

Perishable canned cured meat inoculated with Clostridium botulinum spores was placed at 4.4 or 10 degrees C after manufacture. Spore germination occurred at 10 degrees C. The germinated cell count remained stable over a period of 16 to 18 weeks. During that time period the inhibitory system and residual nitrite descreased. These factors combine to make perishable canned cured meats more prone to spoilage and potential hazard if they are temperature abused after a period of refrigerated storage.     

Effect of Temperature on Adhesion of Vibrio Strain AK-1 to Oculina patagonica and on Coral Bleaching

Laboratory aquarium experiments demonstrated that Vibrio strain AK-1 caused rapid and extensive bleaching of the coral Oculina patagonica at 29 degrees C, slower and less-complete bleaching at 23 degrees C, and no bleaching at 16 degrees C. At 29 degrees C, the application of approximately 100 Vibrio strain AK-1 cells directly onto the coral caused 50 and 83% bleaching after 10 and 20 days, respectively. At 16 degrees C, there was no bleaching, even with an initial inoculum of 1.2 x 10 bacteria. To begin to understand the effect of seawater temperature on bleaching of O. patagonica by Vibrio strain AK-1, adhesion of the bacteria to the coral as a function of temperature was studied. Inoculation of 10Vibrio strain AK-1 organisms into flasks containing 20 ml of seawater at 25 degrees C and a fragment of O. patagonica resulted in net levels of bacterial adhesion to the coral of 45, 78, and 84% after 2, 6, and 8 h, respectively. The adhesion was inhibited 65% by 0.001% d-galactose and 94% by 0.001% methyl-beta-d-galactopyranoside (beta-M-Gal). After the incubation of Vibrio strain AK-1 with the coral for 6 h, 42% of the input bacteria were released from the coral with 0.01% beta-M-Gal, compared to less than 0.2% when beta-M-Gal was present during the adhesion step. Adhesion did not occur when Vibrio strain AK-1 was grown at 16 degrees C, regardless of whether the corals were maintained at 16 or 25 degrees C, whereas bacteria grown at 25 degrees C adhered to corals maintained at 16 or 25 degrees C. Bacteria grown at 25 degrees C adhered avidly to Sepharose beads containing covalently bound beta-d-galactopyranoside but failed to bind if grown at 16 degrees C. These data suggest that elevated seawater temperatures may cause coral bleaching by allowing for the expression of adhesin genes of Vibrio strain AK-1.     

Impact of polyunsaturated fatty acid degradation on survival and acidification activity of freeze-dried <Emphasis Type="Italic">Weissella paramesenteroides</Emphasis> LC11 during storage

The impact of polyunsaturated fatty acid (PUFA) degradation on the survival and acidification activity of freeze-dried Weissella paramesenteroides LC11 was investigated over 90-days storage at 4 degrees C or 20 degrees C in vacuum-sealed aluminium foil or glass tubes with two water activities (a(w)=0.11 or 0.23). Colony counts, acidification activity (% lactic acid/g), linoleic/palmitic (18:2/16:0) or linolenic/palmitic (18:3/16:0) ratio by gas chromatography and 18:2 or 18:3 oxylipins by reversed phase-high performance liquid chromatography were determined. The viable cells, acidification activity and 18:2/16:0 or 18:3/16:0 ratio decreased as the storage time increased. The survival, acidification activity and 18:2/16:0 or 18:3/16:0 ratio were greatest for the freeze-dried strain held in vacuum-sealed aluminium foil at 4 degrees C. The 18:2/16:0 or 18:3/16:0 ratio decrease was correlated with the accumulation of 18:2 or 18:3 oxylipins during storage in glass tubes. Hydroperoxy PUFAs, hydroxy PUFAs, divinyl ether PUFAs and oxo PUFAs were the main oxylipins identified. A large decrease in the 18:2/16:0 or 18:3/16:0 ratio and a rapid accumulation of oxylipins during storage might be enough to cause high cell death and loss of metabolic activity. These results provide further experimental support for the hypothesis that lipid oxidation and survival or activity of freeze-dried bacteria might be related.     

Temperature dependent activation of leucocyte populations of rainbow trout, Oncorhynchus mykiss, after intraperitoneal immunisation with Aeromonas salmonicida

The temperature dependence of in vivo activation of rainbow trout Oncorhynchus mykiss, leucocyte populations after intraperitoneal injection (i.p.) of fish with a T-cell independent antigen Aeromonas salmonicida (strain MT423) was investigated using a proliferation assay and flow cytometric analysis with mab specific for trout leucocyte surface markers. In trout kept at 15-17 degrees C a prominent activation of blood and spleen leucocytes was found. Also, drastic changes of the percentage of the leucocyte populations in blood and spleen occurred: the amount of monocytes in the blood increased between day 2 and day 7 post injection (p.i.), whereas in spleen the amount of monocytes stayed at a high level (approximately 35%) after a depression between day 4 and day 7 p.i. The percentage of B-lymphocytes was increased first in spleen and then in blood. The percentage of granulocytes in blood was elevated during the whole experiment compared to control fish. In trout kept at 10-12 degrees C only blood leucocytes showed a weak activation after i.p. injection of A. salmonicida, whereas spleen leucocytes showed nearly no reaction. Only the percentage of granulocytes in the blood (day 2-14 p.i.) and of monocytes in the spleen (day 2 and day 8 p.i.) was changed compared to phosphate buffered saline (PBS)-injected fish. However, the development of A. salmonicida specific antibodies was contrary to the cellular reaction. Whereas antibodies could first be detected after 16-18 days p.i. in both groups the amount of antibodies was significantly higher in sera of trout kept at 10-12 degrees C at day 22 and day 28 p.i. than in sera of trout kept at 15-17 degrees C. These results indicate stronger A. salmonicida induced activation of monocytes, granulocytes and B-lymphocytes at higher temperature. However, the development of a specific antibody response against A. salmonicida seemed to be more effective at lower temperatures.     

Metabolic responses of chicken embryos to graded, prolonged alterations in ambient temperature

1. Chicken embryos aged 12, 16, 18 and 20 (externally pipped) days of incubation were exposed to graded reductions (2 degrees C) in ambient temperature from 38 to 28 degrees C, exposure to each temperature lasting up to 9 hr. 2. Oxygen uptake was measured first at 38 degrees C and then in the quasi-equilibrium state at lowered temperatures. The temperature coefficient (Q10) was calculated for each egg. 3. For mild cooling (32 degrees C), the Q10 in 18-day-old embryos was about 1.5, while 12- and 16-day-old embryos had a Q10 value of about 2, indicating that a feeble homeothermic metabolic response to cooling appears in late prenatal embryos. It became more marked in externally pipped embryos and further augmented in hatchlings.     

Analysis of n-hexane, 2-hexanone, 2,5-hexanedione, and related chemicals by capillary gas chromatography and high-performance liquid chromatography

Analytical methods, using capillary gas chromatography and normal-phase high-performance liquid chromatography, were developed for the analysis of the neurotoxic chemicals n-hexane, 2-hexanone, and 2,5-hexanedione and their suspected metabolites. Two gas chromatographic methods, using a 50-m glass capillary OV 101 column and cyclohexane as an internal standard, were employed. In both methods, the injector and detector temperatures were 220 and 280 degrees C, respectively. In method I the following temperature program was used: isothermic at 50 degrees C for 30 min, followed by a temperature increase of 10 degrees C/min to a final temperature of 180 degrees C, which was then maintained for 7 min. This method was used to analyze the following compounds: n-hexane, 2,5-dimethylfuran, 2-hexanone, 3-hexanone, hexanal, 1-hexanol, 2-hexanol, 3-hexanol, 5-hydroxy-2-hexanone, gamma-valerolactone, 2,5-hexanedione, and 2,5-hexanediol. Method II, which was developed for n-hexane and eight of its more common metabolites, used the following temperature program: isothermic at 70 degrees C for 15 min, followed by a temperature increase of 40 degrees C/min to a final temperature of 220 degrees C, which was maintained for 5 min. A linear relationship between peak area and amount injected was observed over a 100-fold range. The minimum detectable amounts ranged from 0.05 to 1 microgram, depending on the compound. Normal-phase HPLC, using a 5-micron silica cartridge fitted into an RCM-100 radial-compression separation system, was utilized to analyze 2-hexanone and its metabolites 2,5-dimethylfuran, gamma-valerolactone, 5-hydroxy-2-hexanone, and 2,5-hexanedione.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term survival of shiga toxin-producing Escherichia coli O26, O111, and O157 in bovine feces.

Cattle are an important reservoir of Shiga toxin-producing Escherichia coli (STEC) O26, O111, and O157. The fate of these pathogens in bovine feces at 5, 15, and 25 degrees C was examined. The feces of a cow naturally infected with STEC O26:H11 and two STEC-free cows were studied. STEC O26, O111, and O157 were inoculated into bovine feces at 10(1), 10(3), and 10(5) CFU/g. All three pathogens survived at 5 and 25 degrees C for 1 to 4 weeks and at 15 degrees C for 1 to 8 weeks when inoculated at the low concentration. On samples inoculated with the middle and high concentrations, O26, O111, and O157 survived at 25 degrees C for 3 to 12 weeks, at 15 degrees C for 1 to 18 weeks, and at 5 degrees C for 2 to 14 weeks, respectively. Therefore, these pathogens can survive in feces for a long time, especially at 15 degrees C. The surprising long-term survival of STEC O26, O111, and O157 in bovine feces shows that such feces are a potential vehicle for transmitting not only O157 but also O26 and O111 to cattle, food, and the environment. Appropriate handling of bovine feces is emphasized.     

Comparisons of tumor suppressor p53, p21, and p16 gene therapy effects on glioblastoma tumorigenicity in situ

The mutation and/or deletion of tumor suppressor genes have been postulated to play a major role in the genesis and the progression of gliomas. In this study, the functional expression and efficacy in tumor suppression of 3 tumor suppressor genes (p53, p21, and p16) were tested and compared in a rat GBM cell line (RT-2) after retrovirus mediated gene delivery in vitro and in vivo. Significant reductions in tumor cell growth rate were found in p16 and p21 infected cells (60 +/- 12% vs 66 +/- 15%) compared to p53 (35 +/- 9%). In vitro colony formation assay also showed significant reductions after p16 and p21 gene delivery (98 +/- 5% vs 91 +/- 10%) compared to p53 (50 +/- 18%). In addition, the tumor suppression efficacy were investigated and compared in vivo. Retroviral mediated p16 and p21 gene deliveries in glioblastomas resulted in more than 90% reductions in tumor growth (92 +/- 26% vs 90 +/- 22%) compared to p53 (62 +/- 18%). Tumor suppressor gene insertions in situ further prolonged animal survival. Overall p16 and p21 genes showed more powerful tumor suppressor effects than p53. The results were not surprising, as p16 and p21 are more downstream in the cell cycle regulatory pathway compared to p53. Moreover, the mechanism involved in each of their suppressor effects is different. This study demonstrates the feasibility of using tumor suppressor genes in regulating the growth of glioma in vitro and in situ.