Growth Kinetics, Cell Shape, and the Cytoskeleton of Primary Astrocyte Cultures

Abstract: We examined correlations among growth kinetics, cell shape, and cytoskeletal protein content in rat astrocytes grown in primary culture. Cell suspensions from brains of newborn rats were seeded at densities from 0.2 to 3 × 10⁵/cm². At initial densities above 1 × 10⁵ the population increased to reach confluency by 10–12 days, after which cell number remained stable for many weeks. At low initial densities, 0.2–0.4 × 10⁵/cm², cells did not increase in number. Final density increased with increasing plating densities. High-density cells had small perikarya and several long cytoplasmic processes; low-density cells appeared flat and polygonal. All cultures were almost entirely astrocytic, as judged by immunofluorescent staining with antiserum against glial fibrillary acidic protein (GFAP). Cytoskeletal proteins were analyzed by gel electrophoresis after extraction from cells with nonionic detergent. Relative amounts of the proteins differed, in that low-density cells contained large amounts of cytoskeletal actin relative to the intermediate filament (IF) proteins vimentin and GFAP, whereas high-density cells contained relatively less actin and more IF proteins. Such differences in cytoskeletal proteins between the high- and low-density cultures were mirrored in the relative rates of synthesis of the cytoskeletal proteins. In the low-density cells amino acid incorporation into cytoskeletal-associated actin was more active than that into the IFs, whereas in the high-density cells higher rates of IF protein synthesis were observed.     

Effect of melatonin infusion duration and frequency on gonad, lipid, and body mass in pinealectomized male Siberian hamsters

The goal of this study was to discriminate between two hypotheses regarding how the circadian rhythm of pineal melatonin (MEL) production transmits photoperiodic information: (1) A circadian rhythm of sensitivity to MEL regulates the hormone's effect; (2) the duration of the MEL signal, rather than its circadian timing, is the critical parameter of the MEL rhythm. The experiment examined the response of pinealectomized (PINX) male Siberian hamsters to 10-hr (short-day-type) versus 6-hr (long-day-type) duration MEL infusions (10 ng/infusion) in cycles with period lengths (T) of 18, 24, 36, and 48 hr. After cannula implantation, animals were moved from LD 16:8 to LD 10:14 (lights-on from 0500 to 1500 hr, EST), where the timed infusions began. Additional T 24 cycles included as controls employed 18-hr MEL, 18-hr saline (SAL), and 10-hr SAL infusions: Body weight and food intake were measured weekly. After 6 weeks, animals were killed; blood samples were taken for radioimmunoassay (RIA) of serum follicle-stimulating hormone (FSH) and prolactin (PRL); and terminal body, epididymal white adipose tissue (EPIWAT), and paired testis weights were recorded. Six-hour MEL infusions failed to induce short-day-type effects, regardless of the period (T) of the infusion cycle. In contrast, compared to SAL and 6-hr MEL infusions, 10-hr MEL resulted in decreases in body, EPIWAT, and testis weights in T 24, but not in T 36 or T 48. In T 18, testis, body, and EPIWAT mass were decreased, but not to the same extent as in T 24. Similarly, daily 18-hr MEL infusions (T24) were less effective as a short-day stimulus than were 10-hr MEL infusions. The effectiveness of 10-hr, but not 6-hr, MEL infusions in T 18 and T 24 is consistent with the duration hypothesis and argues against the circadian hypothesis. Neither hypothesis could have predicted that all infusion cycles of T greater than or equal to 36 hr, regardless of the infusion durations, would fail to elicit short-day-type responses. This outcome suggests a need for relatively frequent (T less than 36 hr) MEL stimulation in addition to the requirement for adequate duration of each MEL infusion.     

Lamin dimers. Presence in the nuclear lamina of surf clam oocytes and release during nuclear envelope breakdown

The nuclear lamina of surf clam oocytes contains dimers of 67-kDa lamin which are stabilized by both noncovalent interactions and disulfide bonds. The latter can be reduced but re-form when the reducing agent is removed. The cysteine residues involved in these disulfide bonds are inaccessible to alkylating agents unless the protein is unfolded in urea. During nuclear envelope breakdown the lamin is released as a mixture of oligomers in which disulfide-stabilized dimers are associated noncovalently with lamin monomers. Concurrent with solubilization, both dimers and monomers are phosphorylated to a similar extent, indicating that the interactions which maintain these complexes are not destabilized by lamin phosphorylation. Our results suggest the existence of two types of interactions between the lamin molecules in the polymer, which react differently to phosphorylation during nuclear envelope breakdown.     

Neutrophil adherence receptors (CD 18) in ischemia. Dissociation between quantitative cell surface expression and diapedesis mediated by leukotriene B4

Neutrophils and eicosanoid chemoattractants are centrally involved with ischemia-reperfusion (I/R) injury. The CD 18 complex of adhesive glycoproteins, readily up-regulated by chemoattractants in vitro, is required for polymorphonuclear leukocyte (PMN) adherence to endothelium. This study tests whether CD 18 is up-regulated by ischemia in vivo and its role in mediating PMN diapedesis. Anesthetized rabbits underwent 3 h of bilateral hindlimb tourniquet ischemia (n = 16). Ten min after tourniquet release, levels of plasma leukotriene (LT)B4 increased to 390 +/- 62 pg/ml (mean +/- SE), higher than 134 +/- 26 pg/ml in control rabbits (n = 13, p less than 0.01). Aliquots of plasma were added to whole blood from normal rabbits (n = 6) for flow cytometric analysis of neutrophils with the CD 18 mAb R 15.7. Addition of I/R plasma failed to demonstrate an increase in surface expression of CD 18. Similarly, no CD 18 up-regulation was observed in vivo upon reperfusion in ischemic animals pretreated with mAb R 15.7 (n = 3). However, I/R plasma when introduced into plastic chambers taped atop dermabrasion sites in normal rabbits (n = 12) resulted in diapedesis, measured by the accumulation after 3 h of 1130 +/- 125 PMN/mm3 in the chambers relative to 120 +/- 31 PMN/mm3 with control plasma (p less than 0.01). Diapedesis in response to I/R plasma was abolished by pretreatment with mAb R 15.7 (less than 5 PMN/mm3, n = 6), was reduced by U 75,302, an LTB4 receptor antagonist (253 +/- 101 PMN/mm3, n = 6) (both p less than 0.01) and was not protein synthesis dependent. These results demonstrate that PMN diapedesis in response to I/R plasma is exclusively dependent upon the CD 18 glycoprotein complex by an LTB4-dependent mechanism, despite the fact that CD 18 is not up-regulated on circulating PMN in ischemia. These data indirectly indicate the functional importance of conformational changes of CD 18 in determining PMN adhesion.     

MINIMAL MALE/FEMALE TRADEOFFS IN ZIZANIA PALUSTRIS,A MONOECIOUS ANNUAL GRASS

Because a male/female resource tradeoff is a basic assumption of many models of sex allocation in cosexual plants, statistical and manipulative methods were used to look for evidence of intersexual resource conflicts in Zizania palustris. In this monoecious grass, male and female investments overlap in time within each panicle and on successive panicles, and sex allocation quickly responds to environmental changes. Nevertheless, no negative correlations were found between the numbers of florets of each sex within panicles, on consecutive panicles, or on whole plants. Removing immature fruits or florets of either sex did not significantly increase subsequent investment in the other sex. The one significant tradeoff was slightly lower total fruit production on plants with exceptionally large male investment. Wild rice, therefore, fits the tradeoff assumption of sex allocation models at the population level but rarely at the individual level.