Evidence for regulation of actin synthesis in cytochalasin D-treated HEp-2 cells

In HEp-2 cells treated with 0.2 or 2.0 μM cytochalasin D (CD), the relative rate of actin synthesis increased for about 12 h and then reached a plateau; this increase was suppressed by actinomycin D (AD). When CD was washed from cells which had been treated for 20 h, the elevated rate of actin synthesis declined to the control value within ca 4 h, as the actin-containing cytoskeletal components rearranged by CD recovered their normal morphology. Subsequently, actin synthesis was depressed below control values for a prolonged period; during recovery from 2 h treatment with CD, this depression was of much shorter duration. Re-addition of CD to cells after a 3 h recovery period again induced the cytoskeletal alterations characteristic of CD treatment but did not reverse the prior decline in the rate of actin synthesis. In HEp-2 cells treated with cycloheximide during exposure to CD for 20 h, the relative rate of actin synthesis measured after removal of cycloheximide was twofold higher than with CD alone and such cells exhibited a twofold slower decline in the rate of actin synthesis during recovery from CD in the continued presence of cycloheximide. These effects of cycloheximide, which resemble observations on “super-induction”, suggest that actin synthesis in CD-treated and recovering HEp-2 cells may be regulated by a repressor protein. The possibility that the proposed repressor protein is actin and that actin may thus be a feedback inhibitor of its own synthesis is discussed.     

Effects of short-term cold exposure on pineal biosynthetic function in rats

In light of recent studies demonstrating stress-induced changes in pineal indoleamine metabolism, we tested the effect of acute cold stress on pineal biosynthetic function. Adult male rats were subjected to 30, 60, or 120 min of cold exposure (Ta = 2 degrees C) during either the light or dark phase of the daily photoperiodic cycle. Controls were kept at room temperature (22 +/- 2 degrees C). Animals were killed by decapitation and pineals were analyzed by radioimmunoassay for melatonin content and by radioenzymeassay for the activity of N-acetyltransferase (NAT). Cold exposure during the day elicited no significant changes in pineal indoleamine metabolism. Exposure to cold for 1 hr during the second hour after lights off slightly increased pineal melatonin content, without a concomitant change in NAT activity. Rats exposed to 2 hr of cold beginning 2 hr after lights off, however, displayed a 50% reduction in NAT activity, whereas pineal melatonin content remained unchanged. The paradoxical response of pineal NAT activity and melatonin content are not uncommon when rats are exposed to adverse stimuli.     

Antibody and immune complexes induce tissue factor production by human endothelial cells

Patients with systemic lupus erythematosus (SLE) have an increased incidence of arterial and venous thromboses. The mechanism by which thromboses develop in these patients is unknown. We had previously observed that the sera of patients with SLE contain antibodies and immune complexes that can bind to endothelial cells. Because endothelial cells can synthesize tissue factor, a potent activator of coagulation, we studied the effect of IgG complexes and sera from patients with SLE on the production of tissue factor by these cells. Human umbilical venous endothelial cells incubated with heat-aggregated IgG (HA-IgG) (0.5 to 4.0 mg) elaborate procoagulant activity in a dose-dependent manner. All procoagulant activity was found in the particulate cell fraction, and none was secreted into the medium. Maximum expression of procoagulant activity required 6 to 8 hr, and its production was totally inhibited by the addition of cyclohexamide or actinomycin D. The presence of gel-filtered platelets augmented production of procoagulant activity by endothelial cells stimulated by HA-IgG. Endothelial cell procoagulant activity was not inactivated by diisofluoropropylphosphate, required the presence of Factor VII for its expression, and was neutralized by a specific anti-tissue factor antibody. Endothelial cells incubated with sera from 14 of 16 patients with SLE produced increased amounts of tissue factor compared with 21 normal sera (p less than 0.025). Fractions of two SLE sera containing monomeric IgG, IgA, or IgM, as well as fractions containing IgG complexes, each stimulated endothelial cells to produce more tissue factor than similar fractions prepared from two normal sera. These studies demonstrate that endothelial cells will produce the procoagulant tissue factor after exposure to anti-endothelial cell antibodies or IgG-containing immune complexes. The production of tissue factor by endothelial cells at sites of immune vascular injury may play a role in the development of thromboses in patients with SLE.     

The influence of environmental conditions on the macromolecular composition of Candida utilis

Glucose-limited chemostat cultures of Candida utilis were cultivated at various pH levels (3.0–7.5), temperatures (15–37.5°C), dilution rates (0.06–0.42 hr^?1), and with different nitrogen sources (NH and NO). The ratio of total nucleic acid to protein increased with increase in dilution rate at constant temperature and decreased with increase in temperature at constant dilution rate. The pattern of these variations is consistent with the hypothesis that the nucleic acid to protein ratio is a function of the ratio of the actual dilution rate to the critical dilution rate corresponding to each one of the cultivation temperatures. This ratio is called “reduced dilution rate.” A basis is proposed on which various microorganisms may be compared with respect to the ratios of cell protein to nucleic acid, RNA, ribosomal RNA, and polysomes.     

An isoelectric focusing study of plasma membrane actin.

Plasma membranes were purified from secondary chick embryo fibroblasts labeled with [³⁵S]methionine for 1 or 18 h. The total cell homogenate, postnuclear supernatant and plasma membrane fractions were analyzed by two-dimensional electrophoresis (isoelectric focusing followed by SDS-slab gel electrophoresis). The α, β, and γ isoelectric variants of actin were present in similar proportion in membranes, supernatant, and cell homogenate as determined by incorporation of ³⁵S into each species of actin. These results indicate that the plasma membrane actin of chick fibroblasts is heterogeneous and that no isoelectric variant of actin is unique to the plasma membrane.     

The correlation between specific protein synthesis and tumoricidal function in murine peritoneal macrophages

Macrophages from the lipopolysaccharide (LPS)-responsive C3H/HeN mouse strain and the closely related LPS-nonresponsive C3H/HeJ strain were compared for tumoricidal activation and protein synthetic changes following in vivo and in vitro stimulation, utilizing two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins. Peritoneal macrophages elicited from C3H/HeN mice with heat-killed Propionibacterium acnes exhibited tumoricidal activity in a 16-hr cytolytic assay and expressed cytoplasmic levels of a 23.5-kDa protein during 48 hr of culture. The inability to detect persistent expression of p23.5 in P. acnes-stimulated C3H/HeJ macrophages correlated with the cytolytic impotence of those cells in the 16-hr chromium release assay. C3H/HeN macrophage populations lacking tumoricidal capacity could be rendered lytic, as could P. acnes-elicited C3H/HeJ macrophages, following in vitro stimulation with bacterial lipopolysaccharide. Concomitant with the LPS-induced expression of new functional activity was the appearance of augmented levels of several macrophage-specific proteins, including p23.5. This effect was dependent upon the lipid A moiety of LPS as the effects of LPS could be blocked by inclusion of polymyxin B sulfate in the culture medium. However, neither tumoricidal function nor protein modulation could be readily induced in C3H/HeJ proteose peptone-elicited or resident macrophages. These results identify biochemical responses to stimuli which may be requisite to acquisition or execution of cytolytic activity.     

Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+/K+ homeostasis

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na⁺] and [K⁺] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na⁺‐selective class I‐HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single‐nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35 kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near‐isogenic lines (NILs): 157‐14 (double homozygote for the cheesmaniae alleles) and 157‐17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157‐14 in comparison with 157‐17. A significant difference between NILs was also found for [K⁺] and the [Na⁺]/[K⁺] ratio in leaf and stem but not for [Na⁺] arising a disagreement with the corresponding RIL population. Their association with leaf [Na⁺] and salt tolerance in tomato is also discussed.     

Measurement of cellular DNA mass by flow microfluorometry with use of a biological internal standard

Use of a biological standard (chicken erythrocytes) mixed with experimental cell populations allows control of all variables in flow microfluorometric determination of DNA content. These variables include both staining and instrument procedures. In addition, the use of a biological standard allows determination of cellular DNA mass in unperturbed cell populations. DNA mass measured by FMF technique correlates closely with values reported in the literature that used biochemical techniques.     

Innovative levers for sustainable integration of gender medicine into medical school curricula

Efforts to integrate gender medicine into medical school curricula have focused largely on the work of individual champions. Online sex and gender materials for undergraduate courses have also been developed and disseminated. Success has been sporadic, with varying uptake across schools within and between countries. International trends in medical school accreditation processes and the growing force of the millennial student voice offer untapped opportunities to promote more systematic integration of gender medicine on a national and international level. In this commentary, the president and CEO of the Association of Faculties of Medicine of Canada and the Scientific Director of the Institute of Gender and Health of the Canadian Institutes of Health Research jointly reflect on top-down and bottom-up levers for sustainable innovation in gender medicine for undergraduate medical training.