The nerve growth factor-responsive PC12 cell line does not express the Myc dimerization partner Max.

Heterodimerization of Max with the nuclear oncoprotein Myc and the differentiation-associated proteins Mad and Mxi1 enables these factors to bind E-box sites in DNA and control genes implicated in cell proliferation and differentiation. We show that in the PC12 pheochromocytoma tumor cell line, functional Max protein is not expressed because of the synthesis of a mutant max transcript. This transcript encodes a protein incapable of homo- or heterodimerization. Furthermore, the mutant Max protein, unlike wild-type Max, is incapable of repressing transcription from an E-box element. Synthesis of mutant max transcripts appears to be due to a homozygous chromosomal alteration within the max gene. Reintroduction of max into PC12 cells results in repression of E-box-dependent transcription and a reduction in growth rate, which may explain the loss of Max expression either during the growth of the pheochromocytoma or in subsequent passage of the PC12 cell line in vitro. Finally, the ability of these cells to divide, differentiate, and apoptose in the absence of Max demonstrates for the first time that these processes can occur via Max- and possibly Myc-independent mechanisms.

Relationship between the displacement of phosphatidate phosphohydrolase from the membrane-associated compartment by chlorpromazine and the inhibition of the synthesis of triacylglycerol and phosphatidylcholine in rat hepatocytes.

Glycerolipid synthesis was studied in isolated hepatocytes by using 177 microM [14C]oleate and 1 mM [3H]glycerol. Chlorpromazine (25-400 microM) inhibited the synthesis of phosphatidylcholine and triacylglycerol. This was accompanied by an average increase of 12-fold in the accumulation of the labelled precursors in phosphatidate at 200 microM chlorpromazine and a decrease in the conversion of phosphatidate to diacylglycerol of 76%. These results indicate that part of the inhibition of the synthesis of phosphatidylcholine and triacylglycerol occurs at the level of phosphatidate phosphohydrolase. The relative rate of triacylglycerol synthesis at different concentrations of chlorpromazine was approximately proportional to the rate of conversion of phosphatidate to diacylglycerol. Phosphatidylcholine synthesis increased at higher rates of conversion of phosphatidate to diacylglycerol, but it was relatively independent of the latter rate when this was inhibited by more than about 30% with chlorpromazine. The addition of oleate to the hepatocytes caused a translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Chlorpromazine had the opposite effect and displaced the phosphohydrolase from the membranes in the presence or absence of oleate. There was a highly significant correlation between the activity of phosphatidate phosphohydrolase that was associated with the membranes of the hepatocytes and the calculated conversion of [3H]phosphatidate to diacylglycerol. Chlorpromazine also antagonized the association of the phosphohydrolase with microsomal membranes when cell-free preparations were incubated with combinations of oleate and spermine. Furthermore, it inhibited the transfer of the soluble phosphohydrolase to microsomal membranes that were labelled with [14C]phosphatidate and thereby decreased diacylglycerol production. It is concluded that part of the action of chlorpromazine in inhibiting the synthesis of triacylglycerol and phosphatidylcholine occurs because it prevents the interaction of the soluble phosphatidate phosphohydrolase with the membranes on which glycerolipid synthesis occurs. This in turn prevents the conversion of phosphatidate to diacylglycerol.

Spermine promotes the translocation of phosphatidate phosphohydrolase from the cytosol to the microsomal fraction of rat liver and it enhances the effects of oleate in this respect.

Spermine (0.5-2 mM) promoted the translocation of phosphatidate phosphohydrolase from the soluble to the microsomal fraction in a cell-free system derived from rat liver. By contrast, spermidine (1 mM) and putrescine (1 mM) had no significant effect on the translocation when added alone. Spermine, and to a lesser extent, spermidine, enhanced the translocating action of oleate and increased its effectiveness in transferring the phosphohydrolase from the soluble to the microsomal fraction. It is proposed that the phosphohydrolase becomes metabolically active when it combines with membranes and that polyamines might help to regulate this interaction. This could facilitate the action of fatty acids and enable cells to increase their capacity for triacylglycerol synthesis to match an increased availability of fatty acids.

Long-chain fatty acids and their acyl-CoA esters cause the translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction of rat liver.

A translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction was promoted in cell-free extracts of rat liver by oleate and palmitate and their CoA esters. Oleate was more potent in this respect than palmitate and the CoA esters were more effective than the unesterified acids. Octanoate, octanoyl-CoA and CoA did not cause the translocation. It is proposed that the interaction of phosphatidate phosphohydrolase with the membranes that synthesize glycerolipids causes it to become metabolically active. This enables the liver to increase its capacity for triacylglycerol synthesis in response to an increased supply of fatty acids.

Ciprofloxacin and the fluoroquinolones. New concepts on the mechanism of action and resistance.

Ciprofloxacin, a new fluoroquinolone, is a potent, broad-spectrum antibacterial agent. It rapidly blocks bacterial deoxyribonucleic acid (DNA) replication by inhibiting DNA gyrase, an essential prokaryotic enzyme that catalyzes chromosomal DNA supercoiling. Molecular genetic approaches have been used to study the interaction of 4-quinolones with DNA gyrase from quinolone-sensitive strains and from uropathogenic quinolone-resistant clinical isolates of Escherichia coli. An important mutational locus in the gyrase A gene that confers resistance to ciprofloxacin and other quinolones has been identified, and a new, rapid method to examine clinical isolates for the presence of mutations at this position has been devised. A quinolone resistant gyrA gene has been previously cloned and sequenced from an E. coli clinical isolate. Genetic analysis indicated that resistance resulted from a Ser-83----Trp change in the 875 residue gyrase A protein: two other changes observed in the protein, Asp-678----Glu and Ala-828----Ser, were neutral. GyrA genes carrying these mutations have now been expressed, corresponding mutant gyrase A proteins purified, and their quinolone resistance properties tested by complementing with gyrase B protein and studying the resulting gyrase activity in an adenosine triphosphate-dependent DNA supercoiling assay. The in vitro DNA supercoiling activity of the A (Ser-83----Trp) mutant subunit complemented with wild-type gyrase B subunit was highly resistant to ciprofloxacin and other 4-quinolones. In contrast, A subunit carrying codon 678 and 828 changes reconstituted a quinolone-sensitive gyrase activity. Thus, quinolone-resistant gyrase A proteins may be readily obtained for study by using high-copy gyrA plasmids. In addition, other quinolone-resistant strains of E. coli have been examined for the presence of mutations at gyrase A codons 82 and 83 using a new analytical method based on a restriction fragment length polymorphism (RFLP). This analysis revealed that seven of eight resistant clinical isolates of E. coli examined carried gyrA mutations at codon 82 or 83, whereas five sensitive strains appeared to possess wild-type sequence. Thus, mutations at codon 83 (and possibly 82) in the gyrA gene frequently confer resistance to 4-quinolones, including ciprofloxacin. The RFLP method described should prove useful in examining strains for such mutations. These results are discussed with regard to the mode of interaction of the 4-quinolones with gyrase.

Effects of an antitumoural rhodium complex on thioacetamide-induced liver tumor in rats. Changes in the activities of ornithine decarboxylase, tyrosine aminotransferase and of enzymes involved in fatty acid and glycerolipid synthesis.

Rats were injected daily for 8 weeks with 50 mg of thioacetamide per kg to produce liver tumours. Some of these rats were given three doses of 50 mg of an antitumoural Rh(III) complex/kg at 14, 9 and 5 days before the end of the thioacetamide treatment. Thioacetamide decreased the rate of weight gain of the rats and the Rh(III) complex partly restored it. The activities of ATP citrate lyase, acetyl-CoA carboxylase and fatty acid synthetase in the livers were decreased by thioacetamide treatment and the Rh(III) complex partly reversed this effect. By contrast the activity of malic enzyme was increased by both thioacetamide and the Rh(III) complex and this effect probably relates to NADPH production for detoxification rather than for lipogenesis. Treatment with thioacetamide increased the rate of synthesis of di- and triacylglycerols from glycerol phosphate by liver homogenates, the activity of phosphatidate phosphohydrolase and the incorporation of [3H]glycerol into liver triacylglycerol in vivo. The Rh(III) complex did not produce a significant reversal of these effects of thioacetamide on glycerolipid synthesis. The total uptake of intraportally injected [3H]glycerol by the livers of thioacetamide treated rats was decreased and this was associated with a lowered activity of glycerol kinase. Thioacetamide increased the activity of hepatic ornithine decarboxylase by about 40-fold, but the Rh(III) complex did not reverse this effect. However, the decrease in tyrosine aminotransferase activity that was produced by thioacetamide was partly reversed by the Rh(III) complex. These results are discussed in relation to the tumour-promoting effects of thioacetamide and the antitumoural action of the Rh(III) complex.

Effect of step length optimization on the aerobic demand of running.

To assess whether distance runners displaying uneconomical freely chosen step lengths (FCSL) could be trained to shift FCSL toward a more optimal setting, six males and three females who exhibited uneconomical FCSL [mean optimal step length (OSL) = -9.81% of leg length from FCSL; mean change in oxygen uptake (VO2) (FCSL - OSL) = 1.46 ml.kg-1.min-1] comprised an experimental group that completed 15 treadmill sessions (30 min/day, 5 days/wk, 3 wk) of OSL training at individually determined running velocities (2.87-3.74 m/s). Training sessions featured alternating 5-min periods of combined audio and visual feedback matching OSL and no feedback. A control group of three subjects with uneconomical FCSL (2 males, 1 female) performed 3 wk of treadmill running without feedback. The extent of step length optimization was evaluated by comparing pre- and posttraining differences between FCSL and OSL and between pre- and posttraining VO2. Compared with the control group, the experimental group demonstrated a significantly (P < or = 0.05) greater relative shift in FCSL toward OSL and a marked reduction in FCSL VO2. Taken together, these results suggest that short-term audiovisual feedback training can be effective in optimizing step length and producing a decrease in aerobic demand among distance runners exhibiting uneconomical FCSL.

The association between flexibility and running economy in sub-elite male distance runners.

The purpose of this study was to examine the association between nine measures of limb and trunk flexibility and running economy. Within a week prior to running economy assessment, and after 10 min of jogging at 3.13 m.s-1, 19 well-trained male sub-elite distance runners underwent two complete sets of lower limb and trunk flexibility assessments. Runners then completed two 10-min running economy assessment sessions on consecutive days at 4.13 m.s-1 following two 30-min sessions of treadmill accommodation at 4.13 m.s-1. Intraclass correlation coefficients indicated that the repeated flexibility measurements were highly reliable (X R = 0.92 +/- 0.09), as were the two running economy appraisals (R = 0.99). Correlational analyses revealed that dorsiflexion (r = 0.65) and standing hip rotation (r = 0.53) were significantly (P < or = 0.05) associated with the mean aerobic demand of running, such that runners who were less flexible on these measures were more economical. Although speculative, these results suggest that inflexibility in certain areas of the musculoskeletal system may enhance running economy in sub-elite male runners by increasing storage and return of elastic energy and minimizing the need for muscle-stabilizing activity.

Exercise program effects on women with fibromyalgia syndrome.

The purpose of this study (evaluation) was to examine the effects of an exercise program on 13 women with physician-diagnosed fibromyalgia syndrome (FMS). Participants engaged in exercise for 60 minutes each session. Group 1 (N=7) was in a 3-day-per-week program for 12 months, and group 2 (N= 6) was in a 3-day-per-week program for six months. Group 3 (N= 3) consisted of three participants from Group 1 who participated for six additional months past the 12-month period (total--18 months). Group 3 attended five sessions per week during the six additional months. All participants engaged in aerobic and resistance training. Information was collected on physical fitness, psychosocial, and FMS symptom variables. A majority of the participants appeared to experience a positive outcome on numerous measures of physical fitness, psychosocial factors, and FMS symptoms. Interview data support results. The 13 participants gained various benefits from the exercise program and functioned the same or better outside of the program. Implications for advising FMS patients relative to exercise are given for clinical nurse specialists.

The effect of three test meals on exercise tolerance of an individual with McArdle's disease.

This study examined the effect of three test meals on exercise tolerance of an individual with McArdle's disease, a myopathy characterized by phosphorylase b deficiency. The subject's exercise tolerance and ability to achieve the second wind, in response to each test meal, was evaluated over a 16-week period using a bicycle ergometer in a double-blind situation. Data were analyzed using one-way analysis of variance. No significant differences were found. It was concluded that a high protein meal, a high polyunsaturated fat meal, and a meal containing MCT oil did not affect the exercise tolerance of this individual compared to the control meal.

Exercise, immunity, acute respiratory infections, and homebound older adults.

Appropriate exercise may enhance immune function and lessen acute upper and lower respiratory tract infection (ULRI) symptoms in older adults. Home health care professionals need to know about this potential exercise effect because increased disease resistance and well-being can have a direct impact on activities of daily living (ADL) and independence. This article discusses recent exercise immunology research results, briefly explains the pathways by which exercise might affect immunity and ULRI, and provides guidance for home health care personnel regarding the delivery of an exercise program for their clients.

Soluble fiber: effect on carbohydrate and lipid metabolism.

Dietary fiber consumption has been associated with a decrease in diabetes and atherosclerotic diseases in population surveys. The contribution of soluble fiber (compared to insoluble fiber) to each of these problems has been investigated in several ways. This paper reviews the role which soluble fiber consumption plays in carbohydrate and lipid metabolism. The effects of specific fibers on glucose and insulin responses are examined; possible mechanisms responsible for the described effects are discussed. Epidemiologic evidence that consumption of foods containing soluble fiber contributes to decreased atherosclerotic disease is reviewed. Studies of administration of soluble fiber to hyperlipidemic males and females and normolipidemic subjects are examined with respect to changes in plasma lipids. Soluble fiber appears to play an important role in preventing and possibly treating diabetes and hyperlipidemia, diseases common to Westernized countries.

DNA cloning and organization of the Staphylococcus aureus gyrA and gyrB genes: close homology among gyrase proteins and implications for 4-quinolone action and resistance.

Staphylococcus aureus gyrA and gyrB genes, which encode the DNA gyrase A and B proteins, have been isolated and found to map contiguously. DNA sequence analysis revealed close homology between the S. aureus gyrase subunits and their counterparts in Bacillus subtilis and Escherichia coli, including several conserved amino acid residues whose substitution in E. coli confers resistance to 4-quinolones. These results are discussed in regard to quinolone resistance mechanisms in S. aureus.

Partial purification and characterization of the soluble phosphatidate phosphohydrolase of rat liver.

A method is described by which the Mg2+-stimulated phosphatidate phosphohydrolase can be purified from the soluble fraction of liver from ethanol-treated rats. The increase in specific activity was about 416-fold. This involved purification by adsorption on calcium phosphate, chromatography on DE-52 DEAE-cellulose, separation on Ultrogel AcA-34 and chromatography on CM-Sepharose 6B. The effects of phosphatidylcholine, phosphatidate and Mg2+, Mn2+ and Zn2+ on the activity are described. Inhibitor studies indicate that the phosphohydrolase contains functional thiol groups and arginine residues.

A comparative study of hepatic and epidermal histidase in the guinea-pig (Cavia porcellus).

Histidine ammonia lyase was purified to homogeneity from guinea-pig liver and epidermis. Both enzymes had similar molecular weights, subunit composition and pH optima. Km values for the two were similar at pH 9.2 but different at pH 7.0. Both enzymes were stimulated by low thiol concentrations and inhibited at higher concentrations, but to different extents. Antibody to the hepatic enzyme showed complete identity against hepatic enzyme but incomplete identity against epidermal enzyme.

Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus encoding the DNA gyrase A and B proteins.

We have determined the nucleotide sequence of a 5.3-kb segment of the Staphylococcus aureus chromosome that includes the gyrA and gyrB genes coding for both subunits of DNA gyrase, the enzyme that catalyzes ATP-dependent DNA supercoiling. The gene order at this locus, dnaA-dnaN-recF-gyrB-gyrA, is similar to that found in the Bacillus subtilis replication origin region. S. aureus recF, gyrB, and gyrA genes are closely spaced, occupy the same reading frame, and may be coordinately expressed. The S. aureus gyrB and gyrA genes encode 640- and 889-residue proteins, respectively, that share strong homology with other bacterial gyrase subunits, notably those from B. subtilis. These results are discussed in regard to the mechanism of DNA gyrase and its role as a target for the 4-quinolones and other antistaphylococcal agents.

Physical activity and condition, dietary habits, and serum lipids in second-year medical students.

Level of physical activity has been found to be an independent risk factor for coronary heart disease. Because lifestyle and dietary habits are frequently established by early adulthood, we examined the physical activity, physical fitness, body composition, plasma lipids, and diets of a group of second-year medical students. Medical students were studied because of the presumption that they were knowledgeable about exercise and appropriate diet and would have future influence on their patients. A questionnaire which assessed physical activity was returned by 69 (89%) of the 80 students. Over 50% reported no hard or very hard physical activity either during the week or on weekends. Three subjects were smokers. Body composition, cardiovascular fitness, and plasma lipids were assessed in 20 subjects selected at random from the 69. Five of the 15 men, but none of the five women, had greater-than-desirable body fat. Cardiovascular fitness was at least average compared to normal values, but three had hypertension at rest and 12 had hypertensive responses to exercise. Seven of the men had LDL cholesterol above 130 mg/dl and three had LDL:HDL ratios greater than 3. There was a positive correlation (r = 0.5, p = 0.02) between hard/very hard activity assessed by questionnaire and VO2max and a negative correlation (r = 0.4, p = 0.05) between VO2max and percent fat. All 20 subjects reported above average to severe amounts of stress. Analysis of a 48-hr diet record of 22 students showed an average consumption of 47% carbohydrates, 17% protein, and 36% fat. The polyunsaturated/saturated ratio was 0.43.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the translocation of phosphatidate phosphohydrolase between the cytosol and the endoplasmic reticulum of rat liver. Effects of unsaturated fatty acids, spermine, nucleotides, albumin and chlorpromazine.

The translocation of phosphatidate phosphohydrolase between the cytosol and the microsomal membranes was investigated by using a cell-free system from rat liver. Linoleate, alpha-linolenate, arachidonate and eicosapentenoate promoted the translocation to membranes with a similar potency to that of oleate. The phosphohydrolase that associated with the membranes in the presence of [14C]oleate or 1mM-spermine coincided on Percoll gradients with the peak of rotenone-insensitive NADH-cytochrome c reductase, and in the former case with a peak of 14C. Microsomal membranes were enriched with the phosphohydrolase activity by incubation with [14C]oleate or spermine and then incubated with albumin. The phosphohydrolase activity was displaced from the membranes by albumin, and this paralleled the removal of [14C]oleate from the membranes when this acid was present. Chlorpromazine also displaced phosphatidate phosphohydrolase from the membranes, but it did not displace [14C]oleate. The effects of spermine in promoting the association of the phosphohydrolase with the membranes was inhibited by ATP, GTP, CTP, AMP and phosphate. ATP at the same concentration did not antagonize the translocating effect of oleate. From these results and previous work, it was concluded that the binding of long-chain fatty acids and their CoA esters to the endoplasmic reticulum acts as a signal for more phosphatidate phosphohydrolase to associate with these membranes and thereby to enhance the synthesis of glycerolipids, especially triacylglycerol. The translocation of the phosphohydrolase probably depends on the increased negative charge on the membranes, which could also be donated by the accumulation of phosphatidate. Chlorpromazine could oppose the translocation by donating a positive charge to the membranes.

Biphasic effect of Max on Myc cotransformation activity and dependence on amino- and carboxy-terminal Max functions.

In Ras cotransformation assays, Max exhibited a biphasic effect on Myc transformation activity. Cotransfection of low levels of Max expression plasmid stimulated Myc transformation activity, but cotransfection of high levels suppressed it. Mutations in the functionally undefined Max amino- and carboxy-terminal regions outside of the B/HLH/LZ motif partly separated these activities, suggesting various modes of Max regulation. We demonstrate that the Max protein is a nuclear protein in vivo and identify a carboxy-terminal region similar to nuclear localization signals whose integrity is necessary for efficient localization. Two mutants that delete amino- or carboxy-terminal consensus signals for casein kinase II (CKII) exhibited altered gel mobility and DNA-binding potential in vitro and showed modified transforming potential in the Ras cotransformation assay, suggesting that CKII or a CKII-related enzyme may regulate Max function in vivo. Our data suggest that both the ratio of Myc/Max hetero-oligomers to Max homo-oligomers and Max-specific regulation can contribute to determining the biological activity of Myc in vivo.