Large-scale rotating filter perfusion system for high-density growth of mammalian suspension cultures

Summary A system has been developed for growth and maintenance of mammalian cells in suspension culture at high density. In principle, the maintenance of constant levels of required nutrients coupled with the removal of toxic cell byproducts can support much higher suspension cell densities than may be obtained in conventional spinners. The system consisted of 4- or 40-liter reaction vessels equipped with a vertically supported rotating cylindrical filter. Agitation was provided by the magnetically driven, rotating filter. Fresh medium was supplied at a rate of 10 to 20 ml/h per 10⁹ cells and the expended medium free of cells was withdrawn through the rotating filter. Both pH and dissolved O₂ and CO₂ were monitored and regulated. Walker 256 carcinosarcoma cells have been grown in these reactors to densities 10-to 30-fold greater than that obtained in Bellco spinners. In addition to high cell densities, the yield of cells per liter of medium used was 2- to 3-fold that obtained in the conventional systems. Both 4-and 40-liter versions of this reactor have been operated without the use of antibiotics. The 40-liter reactor also has been modified for chemostat operation. In a single run, for example, the Walker cell density was maintained between 6 and 10×10⁶ cells/ml with a total yield of 8.7×10¹¹ cells from 360 liters of medium.     

Influence of sialic acid on cell surface properties in I-cell disease fibroblasts

Summary Fibroblasts derived from patients with I-cell disease have been shown to accumulate many natural substrates including a three to fourfold increase in sialic acid content compared to that found in normal fibroblasts. This diverse accumulation of storage material is due to a massive deficiency of multiple lysosomal hydrolases as they are preferentially excreted into the culture fluid. There is evidence that the I-cell plasma membrane itself is abnormal with respect to certain transferase activities and in its sensitivity to freezing and Triton X-100. In this study, we have shown that a neuraminidase-sensitive substrate, and perhaps others in I-cell fibroblasts, contribute to an increased electronegativity of the I-cell fibroblast surface and to the cells' sensitivity to freezing. We also found that neuraminidase treatment of I-cell fibroblasts before preservative freezing in liquid nitrogen enables the cells to adapt more easily to subculture upon thawing. This project was supported in part by National Institutes of Health (NIH) BRSG Grant RR-05493, NIH Grant 1-R01-HD-11453-01-A1, National Science Foundation Grant PCM 77-05733, and Maternal and Child Health Service Project 417. Georgirene D. Vladutiu is the recipient of Research Career Development Award 1K04 HD 00312-01A1 from the National Institutes of Health.     

T cells in a suppressor circuit and non-T: non-B cells bear different 1-J determinants

T cells involved in the generation of suppressor activity bear an I-J-subregion controlled determinant (e. g., J₁) which is distinct from that (e. g., J₁) found on non-T: non-13 accessory cells. T-cell subsets examined include Ly-1 inducer and Ly-1,2 acceptor cells which collaborate to generate suppressor activity in the in vitro sheep red blood cell antibody system. Non-T:non-B accessory cells examined include accessory cells involved in concanavalin-A induced, T-cell proliferative responses and in in vitro antibody responses to sheep red blood cells. These results provide evidence for serologic and genetic complexity of the I-J subregion of the murine H-2 gene complex.     

A non-T: Non-B cell bears I-A,I-E,I-J,and Tla (Qa-1?) determinants

A non-T:non-B accessory cell in peritoneal washout or spleen-cell suspensions facilitates T-cell proliferative responses to the mitogen, concanavalin A. Utilizing monoclonal antibody, we show that this accessory cell bears the same I-A- and I-E-subregion controlled determinants as found on B cells. In addition, the same accessory cell bears a Tla (Qa-1?)-region and an I-J-subregion controlled determinant. This I-J determinant is also present on splenic accessory cells involved in in vitro antibody responses to sheep red blood cells. Data in a companion paper show that not all anti-I-J sera contain antibody reactive with the accessory cell, and suggest that T cells involved in the generation of suppressor activity and accessory cells bear different I-J-subregion controlled determinants.     

Urban Philippine healers and their contrasting clienteles

This paper is concerned with medical pluralism in a Philippine setting. It reports on results of a study of four indigenous healers and their patients in Cebu City. The city is a modern medical center in the Philippines, with more than 500 practicing physicians. But its indigenous healers also treat numerous patients, and many patients utilize both physicians and healers during the course of an illness. Of the four healers discussed in this paper, two had the largest followings of any healers in the city at the time of the study, the other two had very modest practices. Significant social and medical contrasts between the clienteles of these healers are described in the paper, and the implications of these differences are discussed with respect to decisions people make about their health care in an area with diverse medical resourses.     

Catabolic enzyme activities in relation to premigratory fattening and muscle hypertrophy in the gray catbird (Dumetella carolinensis)

Summary The flight muscles of the gray catbird (Dumetella carolinensis) were examined to determine if short term adjustments occur in the activity of key catabolic enzymes during preparation for long distance migration. The aerobic capacity of the pectoralis muscle as indicated by citrate synthase activity (CS) is among the highest reported for skeletal muscle (200 moles [min·g fresh mass]^–1 at 25°C). The mass specific aerobic capacity as indicated by CS activity or cytochromec concentration does not change during premigratory fattening (Fig. 2) or in relation to the muscle hypertrophy that occurs concomitantly. The maintenance of mass specific aerobic capacity indicates that the total aerobic capacity increases in proportion to the increase in muscle size. The augmented potential for total aerobic power output is considered an adaptation to meet the increased power requirements of flight due to the increased body mass. Additionally, the capacity to oxidize fatty acids, as indicated by -hydroxyacyl-CoA dehydrogenase activity, approximately doubles during premigratory fattening (from 35 to 70 moles [min·g fresh mass]^–1 at 25°C; Fig. 1A). This adaptation should favor fatty acid oxidation, thereby sparing carbohydrate and prolonging endurance. The activity of phosphofructokinase, a key glycolytic enzyme, does not change before migration.Abbreviations CPT carnitine palmitoyl transferase - CS citrate synthase - HOAD -hydroxyacyl-CoA-dehydrogenase - PFK phosphofructokinase     

Systemic injections of gastro-intestinal peptides alter behavior in rats

Twenty-four male albino rats were given daily intraperitoneal injections of vasoactive intestinal polypeptide (VIP), motilin, human gastrin I (1–17) or the diluent control vehicle at a dose of 100 μg/kg for four consecutive days and food intake, water intake, body weight, and running wheel activity were determined every 24 hours. Animals injected with motilin or human gastrin I (1–17) exhibited decreased food intake relative to those injected with VIP or diluent, which did not differ from each other, although food intake increased reliably over days. The mean water consumption followed the same pattern as that of food intake. As expected from the above results, VIP produced weight gains as compared with rats injected with motilin or gastrin but not reliably more than after diluent. A reliable effect of trials for weight gain was the greatest on day three. Running wheel activity was not affected by injections of human gastrin I (1–17), motilin, or diluent but was reliably decreased by VIP. No significant differences existed across days. Although the results indicate that GI peptides may affect behavior when injected systemically and that like other peptides they have multiple effects, caution is urged in the interpretation of behavioral results at this time.     

Nasal vasoconstrictor activity of a novel PGE2 analog

A novel PGE₂ analog (CL 116,069) was shown to be effective in dogs as a nasal decongestant. Threshold doses were approximately 0.1 μg/kg with intravenous administration and between 0.08 and 4 μg with topical administration. CL 116,069 was compared to 17-phenyl-trinor PGE₂ (CL 116,147), a compound recently studied in humans, and xylometazoline, a well-known nasal decongestant. When given i.v., efficacious doses of xylometazoline tended to raise blood pressure and be shorter acting than the PGs, which did not affect blood pressure. When given topically, all three were long-acting. CL 116,069 usually had the lowest threshold and CL 116,147 usually induced the smallest response. All three agents were more effective than PGE₁ or PGE₂. A 30-day (b.i.d., topical) toxicity test with CL 116,069 produced no inflammation or nasal pathology and no loss in tissue sensitivity. $\text{In}$$\text{vitro}$ examination of xylometazoline and CL 116,069 for vascoconstrictor activity on dog isolated mucosa revealed a response profile similar to that observed with these agents $\text{in}$$\text{vivo}$; i.e., the magnitude of response was comparable for both agents but the t $\text{1}\text{2}$ was only 74 minutes for xylometazoline and greater that 6.5 hours for CL 116,069. The data suggest that CL 116,069 may provide a therapeutic alternative in which constriction of the nasal blood vessels need not be associated with a generalized vasoconstrictor liability.     

POLLEN MORPHOLOGY OF HAPLOPAPPUS AND RELATED GENERA (COMPOSITAE—ASTEREAE)

Pollen grains of Haplopappus and related genera in the subtribe Solidaginae from North and South America were examined by light and scanning electron microscopy. The grains are consistently tricolporate and echinate. Some genera can be distinguished by pollen size, spine length, and number of spine rows between colpi. Based on these characters, the divergence of Benitoa from other members of the subtribe, as indicated by its morphology and secondary chemistry, is supported. Additionally, the recently suggested absence of a close relationship between Pyrrocoma and Oonopsis is indicated by their contrasting pollen types. This study demonstrates the potential of pollen studies in distinguishing some taxonomic groups in the Astereae.