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.     

Early detection and treatment of hyperlipidemia: physician practices in Canada.

We surveyed primary care physicians in Canada to determine their current practices regarding the detection and treatment of hyperlipidemia in asymptomatic adults 20 years of age or more and to determine the role of selected patient characteristics (age, sex and the presence of coronary heart disease [CHD] risk factors) in their management decisions. The self-administered questionnaire was completed by 428 of 804 family physicians and general practitioners. The proportion of physicians who reported having tested at least 50% of their adult patients varied from 29% to 85% and was related to the number of CHD risk factors present and the patient''s age. The proportion of respondents who reported starting dietary or drug therapy among patients with a cholesterol level of 6.2 mmol/L or less increased as the number of CHD risk factors increased and was not related to patient age or sex. According to the factors examined our results suggest that primary care physicians in Canada select patients for screening and treatment mainly on the basis of CHD risk factors present and that their approach is more conservative than that recommended by the Canadian and US consensus conferences.     

Water Balance in Cucumis Plants, Measured by Nuclear Magnetic Resonance, I

In this paper we demonstrate the study of plant water balanceby the non-invasive measurement of tissue water content andwater flow using proton nuclear magnetic resonance (NMR). Sapvelocity and flux were measured independently in the presenceof an excess of stationary tissue water. The instrumentationdescribed allows automated and unattended measurement of flow-and water content-variables in a well-defined region of theplant over periods of several days, with a time resolution betweensuccessive measurements of c. 5 s. Using this apparatus theeffect of changes in light intensity (day/night rhythm) andrelative humidity on stem tissue water content as well as onthe velocity and flux of xylem sap in the stem were investigatedin a cucumber plant. The results are in agreement with predictionsfrom a simple model for plant water balance, which is basedon water potential, flow rate and resistance to flow. As longas only transpiration is varied, flow rate and water content(or potential) are affected in opposite ways as demonstratedin this paper. In contrast, the model predicts that changesin uptake (resulting from changes in, for example, root resistance)will induce changes in water content and flow in the same direction.An experimental verification of this prediction is given ina subsequent paper, where, in addition, the NMR results arecompared to those obtained with a dendrometer. Key words: Water balance model, Cucumis sativus L., flow, water content, NMR, water balance measurement     

Characterization of lipopolysaccharide-induced macrophage gene expression

A cDNA library from LPS-treated murine peritoneal macrophages has been screened by differential hybridization with radiolabeled cDNA from untreated and LPS-treated macrophages. Six clones hybridizing with mRNA sequences present in LPS-treated cells but not in controls were selected for further characterization. When the recombinant bacteriophage DNA from each clone was used as a probe in Northern analysis of total RNA from LPS-treated macrophages, inducible mRNA ranging from 1.45 to 6.4 kb were seen. In five of six cases, the mRNA expression was undetectable in untreated macrophage cultures. All but one clone identified mRNA that were inducible even in the presence of cycloheximide, indicating the independence of such gene expression from protein synthesis; none of the genes were superinduced by this treatment. The time course of expression differed among the individual genes. Four were induced transiently, whereas two showed stable increasing accumulation through an 8-h period after stimulation. In addition, four of the genes were seen within 30 min of stimulation, whereas two were seen only after 2 to 4 h. Two genes were induced only by treatment with LPS, whereas four were also induced in response to other agents, including IFN-gamma, macrophage CSF, and PMA. The insert sequences from these recombinant clones did not hybridize with a set of cDNA encoding other inducible gene products, including TNF, IL-1, ornithine decarboxylase, c-myc, c-fos, JE, or KC. Thus, these six cDNA appear to encode inducible macrophage genes that are distinct from one another as well as from a selection of previously described early genes. Although their functional identity remains indeterminate, they may encode previously described early proteins induced in macrophages treated with LPS.     

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.     

TASTE GROUP THRESHOLDS AND SENSORY EVALUATION OF SPANISH WINE VINEGARS

Wine vinegar is a product obtained from wine acidification which contains at least 5% by wt. of acetic acid, in general without any additives or colorings.
Aspects studied in this work include: the determination of the taste group thresholds (geometric mean of the individual best-estimate thresholds "BET") of two different acids (citric and acetic acids) in aqueous solution and spanish vinegars produced from table and sherry wines. The results obtained suggest that wine vinegar can be considered something more than just an acidulant agent.
In order to evaluate differences among wine vinegars, discriminant tests for twenty-five spanish vinegars (sherry, table and flavored vinegars) were applied. Six of the twelve attributes freely chosen by assessors allowed grouping of the spanish wine vinegars according to their sensory aspects.     

Arginine deprivation in KB cells: I. Effect on cell cycle progress

When exponentially growing KB cells were deprived of arginine, cell multiplication ceased after 12 h but viability was maintained throughout the experimental period (42-48 h). Although tritiated thymidine ([(3)H]TdR) incorporation into acid-insoluble material declined to 5 percent of the initial rate, the fraction of cells engaged in DNA synthesis, determined by autoradiography, remained constant throughout the starvation period and approximately equal to the synthesizing fraction in exponentially growing controls (40 percent). Continous [(3)H]TdR-labeling indicated that 80 percent of the arginine-starved cells incorporated (3)H at some time during a 48-h deprivation period. Thus, some cells ceased DNA synthesis, whereas some initially nonsynthesizing cells initiated DNA synthesis during starvation. Flow microfluorometric profiles of distribution of cellular DNA contents at the end of the starvation period indicated that essentially no cells had a 4c or G2 complement. If arginine was restored after 30 h of starvation, cultures resumed active, largely asynchronous division after a 16-h lag. Autoradiographs of metaphase figures from cultures continuously labeled with [(3)H]TdR after restoration indicated that all cells in the culture underwent DNA synthesis before dividing. It was concluded that the majority of cells in arginine-starved cultures are arrested in neither a normal G1 nor G2. It is proposed that for an exponential culture, i.e. from most positions in the cell cycle, inhibition of cell growth after arginine with withdrawal centers on the ability of cells to complete replication of their DNA.     

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.