[Determination of sodium phenytoin concentration in the blood by radioimmunologic PEG].
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Biomedical subjects
Publications and source records attributed to T H Chen.
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Gap-junctional intercellular communication is a biological process implicated in the regulation of cell proliferation and differentiation. Metabolic cooperation between 6-thioguanine-sensitive and resistant Chinese hamster cells, in vitro, has been used as a means to detect chemicals which can inhibit this form of intercellular communication. To further characterize this in vitro system as a potential screening assay for potential teratogens, tumor promoters and reproductive toxicants, a series of common solvents as well as other chemicals representing eight different functional groups, i.e., alcohols with straight or side chains, glycols, ketones, esters, ethers, phenols, aldehydes, amines and amino compounds and oxygen-heterocyclic compounds, were tested for their ability to inhibit colony-formation and to inhibit metabolic cooperation. A wide range of effects were observed which suggested a structure/activity relationship between a chemical's ability to inhibit gap junction-mediated intercellular communication and the cytotoxicity of a chemical. Possible mechanisms affecting the modulation of gap junctional communication by these chemicals are discussed.
The freezing behavior of dimethylsulfoxide (DMSO) and sorbitol solutions and periwinkle (Catharanthus roseus) cells treated with DMSO and sorbitol alone and in combination was examined by nuclear magnetic resonance and differential thermal analysis. Incorporation of DMSO or sorbitol into the liquid growth medium had a significant effect in the temperature range for initiation to completion of ice crystallization. Compared to the control, less water crystallized at temperatures below -30 degrees C in DMSO-treated cells. Similar results were obtained with sorbitol-treated cells, except sorbitol had less effect on the amount of water crystallized at temperatures below -25 degrees C. There was a close association between the per cent unfrozen water at -40 degrees C and per cent cell survival after freezing for 1 hour in liquid nitrogen. It appears that, in periwinkle suspension cultures, the amount of liquid water at -40 degrees C is critical for a successful cryopreservation. The combination of DMSO and sorbitol was the most effective in preventing water from freezing. The results obtained may explain the cryoprotective properties of DMSO and sorbitol and why DMSO and sorbitol in combination are more effective as cryoprotectants than when used alone.
A procedure for cryogenic storage of alkaloid producing cell lines of periwinkle, Catharanthus roseus (L.) G. Don., has been developed. The procedure differs from established cryopreservation protocols in several aspects. Specifically, 4-day-old suspension subcultures of three cell lines were precultured in nutrient media supplemented with 1 molar sorbitol for 6 to 20 hours. The cells were then incubated in nutrient media with 1 molar sorbitol plus 5% DMSO in an ice bath for 1 hour and, thereafter, were frozen in this solution at a cooling rate of 0.5 degrees C per minute to -40 degrees C prior to immersion in liquid nitrogen (LN). After rapid thawing in a 40 degrees C water bath, the regrowth of LN stored cells was achieved by transferring them without washing onto filter paper discs over nutrient media solidified with agar for a period of 4 to 5 hours. The filter paper discs with the cells were then transferred to fresh media of the same composition for regrowth. The viability immediately after thawing as evaluated by the 2,3,5-triphenyl tetrazolium chloride method was about 60% of controls. Suspension cultures established from LN stored cells retained the capability for alkaloid synthesis and accumulation.
The freezing of deep undercooled water in cold-hardened 3-year-old stems of 16 woody taxa was studied in mid-January by differential thermal analysis. The initiation temperature and the size of the low temperature exotherm (LTE) were compared for nonthawed, thawed, and freeze-killed stems. In general, the initiation temperature of the LTE for nonthawed stems occurred at a lower temperature than for thawed stems and freeze-killed stems. In some cases, no LTE was detected in nonthawed stems although a LTE was detected after thawing. The size of the LTE increased after thawing the stem and also after the stem was freeze killed. The LTE observed in one species disappeared upon exposure to continuous low sub-zero temperatures. Results suggest that undercooling which subsequently results in the LTE in woody stems is due to the cell wall and the plasma membrane. During periods of prolonged freezing, cellular water migrates from the cells which undercool to extracellular ice. This results in a concentration of cell solutes which lowers the homogeneous nucleation temperature of the cell sap. The cold hardiness of nonthawed and thawed stems was compared by a controlled freeze test. In general, thawing had little effect on the survival temperature whereas it had a marked effect on the initiation of the LTE.
The effect of abscisic acid (ABA) on the cold hardiness of cell suspension was investigated. Cell suspension cultures of winter wheat (Triticum aestivum L. cv Norstar), winter rye (Secale cereale L. cv Cougar), and bromegrass Bromo inermis Leyss treated with 7.5 x 10(-5) molar ABA for 4 days at 20 degrees C could tolerate -30 degrees C, whereas the control cultures tolerated only -7 to -8 degrees C. The optimum concentration for increasing the cold hardiness of the cultures was 7.5 x 10(-5) molar. The degree of cold hardiness and the rate of hardening obtained by ABA treatment was significantly higher than that induced by low temperature alone. Of ten species tested, ABA was only effective on those cultures which were capable of cold hardening upon exposure to low temperatures. The results suggest that ABA bypasses the cold requirement for hardening and also suggests that ABA triggers the genetic system(s) responsible for inducing the hardening process.
Upon exposure to 2 degrees C, the leaves and crowns of rye (Secale cereale L. cv ;Puma') and wheat (Triticum aestivum L. cv ;Norstar' and ;Cappelle') increased in cold hardiness, whereas little change in root cold hardiness was observed. Both root and shoot growth were severely reduced in cold-hardened Norstar wheat plants frozen to -11 degrees C or lower and transplanted to soil. In contrast, shoot growth of plants grown in a nutrient agar medium and subjected to the same hardening and freezing conditions was not affected by freezing temperatures of -20 degrees C while root growth was reduced at -15 degrees C. Thus, it was apparent that lack of root development limited the ability of plants to survive freezing under natural conditions.Generally, the temperatures at which 50% of the plants were killed as determined by the conductivity method were lower than those obtained by regrowth. A simple explanation for this difference is that the majority of cells in the crown are still alive while a small portion of the cells which are critical for regrowth are injured or killed.Suspension cultures of Norstar wheat grown in B-5 liquid medium supplemented with 3 milligrams per liter of 2,4-dichlorophenoxyacetic acid could be cold hardened to the same levels as soil growth plants. These cultures produce roots when transferred to the same growth medium supplemented with a low rate of 2,4-dichlorophenoxyacetic acid (<1 milligram per liter). When frozen to -15 degrees C regrowth of cultures was 50% of the control, whereas the percentage of calli with root development was reduced 50% in cultures frozen to -11 degrees C. These results suggest that freezing affects root morphogenesis rather than just killing the cells responsible for root regeneration.
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A 3-day-old infant, born to a mother with preeclampsia, suffered intrauterine asphyxia and subsequently had multiple erythematous subcutaneous plaques and nodules. The clinical setting, the skin manifestations, and the histologic appearance of the lesions were diagnostic of subcutaneous fat necrosis of the newborn. Transient thrombocytopenia was noted with the appearance of the skin lesions, but returned to normal as the skin lesions spontaneously resolved.
In 1976, in a small, remote Libyan village, one apparently sick camel was slaughtered and skinned, and the camel meat was distributed for human comsumption. A few days later, 15 villagers suffered a severe febrile illness. Of the five individuals who had participated in the killing and dispensation of the camel, all were dead within four days. When samples of serum from nine of the remaining patients were examined, seven were found to be positive for plague as determined by the passive hemagglutination test. Another six persons became ill after killing two goats, and the serum of one goat contained antibodies to Yersinia pestis. Because all of the remaining patients except one were treated early enough, they recovered. These incidents confirm previous reports that the camel and the goat are susceptible to naturally occurring plague infection and have a significant role in the dissemination of human plague.
The effect of dosage and route of inoculation of bacille Calmette-Guérin (BCG) on immune response to allogeneic tumor cells was investigated. BALB/c mice were tested 14 and 21 days after injection of EL-4 lymphoma for spleen-cell cytotoxicity against EL-4 cells in vitro and for complement-dependent, antibody-mediated lysis of tumor cells. BCG treatment had no measurable effect on the antibody-mediated lysis of tumor cells, but spleen-cell cytotoxicity was significantly increased in mice treated with 10(4) or 10(8) BCG by the intraperitoneal route; no such increase occurred when BCG was given by the oral or subcutaneous routes. The cytotoxic effector cells were primarily thymus-derived, since treatment of spleens with rabbit antiserum to mouse brain serum decreased cytotoxicity titers by approximately 90%. Within the framework of these experiments, the intraperitoneal route of BCG inoculation resulted in a more effective immune stimulation than the oral or subcutaneous routes.
Protection against pneumonic plague by the oral administration of a live, attenuated Yersinia pestis vaccine, EV76 (Paris) F, was evaluated in the vervet (Cercopithecus aethips). Six animals were vaccinated with a dose of 1.175 X 10(9) colony-forming units (cfu); all tolerated the dose and developed antibodies to Fraction I of Y. pestis. Three immunized animals were challenged with an inhaled dose of 3.2 X 10(6) cfu (160 LD50 [50% lethal dose]) of virulent Y. pestis strain 195/P and two survived; the other three received a challenge dose of 4.9 X 10(6) cfu (245 LD50) and one survived. The three surviving monkeys had high titers of antibody to Fraction I. At necropsy of the animals that succumbed to the challenge infection, almost complete consolidation of the lungs was apparent, and sizable effusions were present in the thoracic cavities. Indeed, the striking pathology indicated the severest form of pneumonic plague. Since it can be assumed that the animal surviving challenge with the larger inoculum would have survived the smaller dose, we conclude that the immunization procedure protected half of the vervets that were exposed to challenge.
Virulent Yersinia pestis was grown on heart infusion blood agar and examined by scanning electron microscopy, exposing the fraction 1 envelope antigen on cell surfaces as a lumpy coating that spilled into the surrounding milieu. The amount of antigen depended on both the duration and temperature of incubation. Comparison of the cultures grown at 37 degrees C for 24, 48, and 72 h with those grown at 22 degrees C for identical periods demonstrated that the antigen increased in amount with the length of incubation, and that the overall production of antigen was much greater of 37 degrees C than at 22 degrees C. These experiments visually confirmed the findings of our previous immunological studies. Moreover, we established that the closely related, virulent organism Y. pseudotuberculosis bears no such antigenic coating at any temperature or incubation period. In addition, the emergence of multiple flagella was noted when Y. pseudotuberculosis was grown at 22 degrees C in a liquid medium, whereas Y. pestis remained without these organelles. These observations preceptibly corroborated the absence of fraction 1 envelope antigen and the presence of flagella, respectively, for distinguishing Y. pseudotuberculosis from Y. pestis.
A live, attenuated Yersinia pestis vaccine, designated EV76 (Paris) F, was pathogenic in Cercopithecus aethiops when administered parenterally. Although an oral dose of 10(9) colone-forming units of the vaccine was nonfatal to vervets, a transient but severe gastrointestinal disturbance resulted in four of 12 animals. Seven immunized vervets were protected against intradermal challenge. The remaining five vervets without serologic reactions and five untreated controls died from typical bubonic (septicemic) plague within seven days. General reactions after vaccination were not predictive of later-developing immunity, which appeared to correlate with the production of Fraction I-specific antibodies. Marked differences in immunity were noted with two methods of vaccine administration (incorporation into bananas and dropping into the mouth). This finding supported the prior observation that the oral mucosa is the major portal of entry for this strain of vaccine. Local reactions in the buccal cavity after feeding were negligible. Thus this strain of Y. pestis is a nonlethal, effective weapon against bubonic plague in a susceptible species.
In experiments designed for study of bacteremia and serologic response of Macaca cynomolgus philippinensis to vaccination with a soluble antigen (fraction I) of Brucella melitensis and the live Rev. I vaccine strain, fraction I and Rev. I were administered both four weeks apart and simultaneously. One of three monkeys given the vaccines four weeks apart and two of three monkeys vaccinated with fraction I and Rev. I in a single injection developed transient bacteremia, as shown by blood cultures. Both methods of vaccination induced significant titers of antibody to B. melitensis, and vaccinated monkeys were protected against subsequent challenge with a virulent strain of B. melitensis.
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