Characterization of fatty acid synthase in cell lines derived from experimental mammary tumors.

The lipogenic enzyme fatty acid synthase (FAS) is elevated in various human primary cancers and certain human cancer cell lines. FAS overexpression in human neoplasia has clinical relevance because of its association with tumor aggression and potential chemotherapeutic intervention. Here, we surveyed FAS in cell lines established from normal murine mammary epithelium (NMuMG) and from mammary tumors induced by either rodent polyoma (Py) virus or murine mammary tumor virus (MMTV). Western blotting revealed greater content of FAS in Py-transformed A1-1 and T1 than NMuMG or MMTV-transformed Mm5MT, RIIIMT and MMT060562. These data suggest that signaling events mediated by Py transformation may increase cellular amounts of FAS. Although FAS content was elevated to similar levels in A1-1 and T1, specific activities were significantly different as enzyme activity in T1 was 3-fold higher than A1-1. Likewise, FAS activity in NMuMG was about 0.5-fold higher than the MMTV-transformed lines, even though enzyme content was similar. Immunoprecipitation studies employing anti-phosphoamino acid antibodies followed by immunoblot analysis with anti-FAS antisera (and vice versa) were used to characterize the constitutive phosphorylation state of the enzyme. Phosphoserine and phosphothreonine residues were detected in the more active FAS from T1 and NMuMG, but not in the less active FAS from Mm5MT or A1-1. Discovery of phosphorylated FAS suggests that the enzyme may have more immediate control over lipogenesis than previously thought. High-dose (10-4 M) dexamethasone induced FAS content and activity in NMuMG and MMTV-transformed lines but not Py-transformed cells. Lower concentrations (10-8, 10-6 M) of dexamethasone also activated FAS but without concomitant elevation of its protein content, which was consistent with a phosphorylated form of FAS. Finally, cell lines were treated with the FAS inhibitor cerulenin: almost all breast cancer lines were growth inhibited at significantly lower amounts of drug than normal cell lineages, suggesting that FAS plays a greater role in viability of tumor cells than normal cells. Pretreatment with palmitate (a primary end-product of FAS) prior to cerulenin rescued A1-1 cells only slightly from growth inhibition, whereas pretreatment with oleate (a monounsaturated fatty acid synthesized from palmitate) synergized cerulenin's cytotoxic effects.

Increased expression of fatty acid synthase (OA-519) in ovarian neoplasms predicts shorter survival.

Certain cancers exhibit derangement of de novo fatty acid biosynthesis, manifested as overexpression and hyperactivity of the lipogenic enzyme fatty acid synthase (FAS). Correlation of elevated FAS with high tumor grade and advanced stage in primary breast, prostate, and colorectal cancers has drawn attention to the enzyme as a possible marker of poor prognosis. To find a similar utility of FAS in ovarian neoplasms, we compared FAS expression in 68 ovarian tumors with their histological features and clinical outcome. Immunohistochemical localization of FAS was observed in 48 (71%) cases in which staining was either focal (defined as positive staining in 1% to 20% of cells) or multifocal/diffuse (positive staining in >20% of cells). Most (83%) of the 48 cases were represented by endometrioid, serous, or mucinous carcinomas and malignant mixed mullerian tumors (MMMTs). In contrast, ovarian adenomas and tumors of low malignant potential (LMPs) contained little or no FAS. Association between FAS expression and histological diagnosis was statistically significant. The extent of FAS immunostaining was also predictive of prognosis. Among all patients with ovarian malignancies (including LMPs), median survival was 64.8 months, when their tumors exhibited no or focal immunostaining for FAS, as opposed to 31.2 months, when staining was multifocal/diffuse (P = .005). Similar median survival values were obtained when cases were limited to endometrioid, serous, and mucinous carcinomas. Short-term survival at 1 and 2 years was significantly higher in patients whose tumors showed no or focal expression of FAS compared with multifocal/diffuse expression. Thus, elevated FAS may serve as an independent marker for predicting poor clinical outcome in patients with ovarian cancer.

Cryptosporidiosis associated with a swimming pool complex.

Twelve children and one adult in Gloucestershire fell ill with cryptosporidiosis in March 1992. Ten cases lived in or near a particular Cotswold town. Eight of these had visited a swimming pool within 24 hours of a suspected faecal accident, and two may have been secondary cases. Of the three cases who lived elsewhere, one was probably unrelated to the outbreak, one had visited the pool, and one was a contact of a boy who may have been the source of the outbreak.

An outbreak of Legionnaires' disease in Gloucester.

Fourteen people living in or near the city of Gloucester fell ill with Legionnaires' disease caused by Legionella pneumophila serogroup (SG) 1 between 27 August and 27 October 1986. Another patient had fallen ill on 30 May. Nine of the 15 were diagnosed retrospectively during a case finding exercise. There were three deaths. Three cases of Pontiac fever were also diagnosed. The source was probably one or more wet cooling towers. Nineteen premises in the city with such towers were identified, and three just outside Gluocester. Samples from 11 of the 22 premises grew Legionella spp.; from nine of these L. pneumophila SG 1 (Pontiac) was isolated. The efficacy of regular addition of biocide in addition to hypochlorite added at the time of disinfection in inhibiting the growth of Legionella spp. was demonstrated. A survey of patients' movements during their likely incubation period showed that there was no single building that all patients had visited, but there were two areas of the city which nearly all had visited or passed through by car. A case-control study demonstrated an association with one of these areas. Cooling towers near both areas may have been sources but the evidence is insufficient to incriminate any single one. The unexpected finding of L. pneumophila SG 1 (Pontiac) in nine towers supports the hypothesis that there may have been multiple sources. Cooling towers may have been contaminated by mains water or by drift from other towers.

Phylogenetic analysis and evolution of RNase P RNA in proteobacteria.

The secondary structures of the eubacterial RNase P RNAs are being elucidated by a phylogenetic comparative approach. Sequences of genes encoding RNase P RNA from each of the recognized subgroups (alpha, beta, gamma, and delta) of the proteobacteria have now been determined. These sequences allow the refinement, to nearly the base pair level, of the phylogenetic model for RNase P RNA secondary structure. Evolutionary change among the RNase P RNAs was found to occur primarily in four discrete structural domains that are peripheral to a highly conserved core structure. The new sequences were used to examine critically the proposed similarity (C. Guerrier-Takada, N. Lumelsky, and S. Altman, Science 246:1578-1584, 1989) between a portion of RNase P RNA and the "exit site" of the 23S rRNA of Escherichia coli. Phylogenetic comparisons indicate that these sequences are not homologous and that any similarity in the structures is, at best, tenuous.

Effect of vascular cannulation on serum concentrations of LH, FSH, prolactin and cortisol in prepubertal gilts.

Two experiments were conducted to determine whether cannulation of the jugular vein in gilts alters serum concentrations of LH, FSH, prolactin (PRL) or cortisol (C). In Experiment 1, 12 crossbred prepubertal gilts weighing 95 +/- 1.3 kg were immobilized by snaring, and tygon tubing was threaded into the anterior vena cava through a 12-gauge needle inserted into the jugular vein. Five hours later, blood samples were drawn at 20-min intervals for 4 h (Day 0). Samples were also drawn at 20-min intervals for 4-h periods 24 h (Day 1) and 48 h (Day 2) after cannulation. Serum concentrations of LH were similar (P=0.26) among Day 0 (0.40 ng/ml), Day 1 (0.39 ng/ml) and Day 2 (0.34 ng/ml). Serum PRL was similar (P=0.07) among Day 0 (4.10 ng/ml), Day 1 (3.87 ng/ml) and Day 2 (3.43 ng/ml). Serum concentrations of C were greater (P < 0.001) on Day 0 (8.32 ng/ml) than Day 1 (4.48 ng/ml) or Day 2 (3.54 ng/ml). In Experiment 2, cannulas were placed in 29 prepubertal gilts. Two days after initial cannulation, six blood samples were drawn at 20-min intervals. Gilts were then immobilized by snaring, and a second cannulae was inserted into the contralateral vein. Five blood samples were taken at 2-min intervals during the second cannulation and then six samples were drawn at 20-min intervals. Serum LH and FSH were not altered by cannulation or elevated during the subsequent 2-h sampling period (P>0.05). In contrast, serum concentrations of PRL rose slowly (P<0.05) during cannulation and remained elevated for 60 min before returning to baseline. Serum concentrations of C rose within 6 min of cannulation, remained elevated for 30 min, and then declined over the next 90 min. From these two experiments, it appears that secretory patterns of LH and FSH can be accurately assessed immediately after cannulation in prepubertal gilts. Measurements of serum PRL and C that reflect nonstressed conditions, however, cannot be obtained until at least 2 h or 1 d after cannulation, respectively.

Evaluation of an interfragmentary compression system for the repair of equine femoral capital physeal fractures.

Femoral neck and proximal epiphyseal lengths were measured in 37 femurs from 19 cadaver foals that were 1 day to 12 months old to determine the applicability of a human interfragmentary compression system to equine femoral capital physeal fractures. Because components of the implant system are available only in fixed sizes, its use was possible in foals older than 5 weeks of age, but not in younger foals. The 135 degree angle plate conformed best to the equine femur. Femoral capital physeal fractures were created surgically and repaired with the implant system in three foals. Fracture stability was evident clinically and radiographically in all three foals until euthanasia at month 3. At necropsy, the treated femurs were 4, 8, and 27 mm shorter than their mates. Epiphyseal viability was verified in all three foals by tetracycline deposition and new appositional bone growth comparable with that in the contralateral control epiphyses. The treated capital physis was open but reduced in thickness in one foal, disorganized in one foal, and closed in one foal. Fixation by compression with the implant system resulted in stability sufficient for fracture healing and maintenance of epiphyseal viability, although it was associated with reduced longitudinal femoral growth.

Femoral capital physeal fractures in 25 foals.

The medical records of 25 horses 1 year of age or younger affected with femoral head and neck fractures during an 18 year period were reviewed. Each fracture involved the capital physis. The foals were 11 days to 12 months of age (mean, 5 months). No femoral capital physeal fractures occurred in horses older than 1 year of age during the same period. The history in each case included acute onset of severe unilateral hindlimb lameness, 3 hours to 2 months (mean, 12 days) before presentation. Injuries observed were violent falls, struggles, and kicks. Crepitation, swelling, pain with manipulation or palpation or both, and apparent fracture fragment displacement were inconsistently noted. Tentative clinical diagnoses were confirmed by radiography in 24 foals and by necropsy alone in one foal. Twenty-one foals were euthanatized due to poor prognosis. One foal sent home for stall rest was lost to follow-up. Surgical repair was attempted in three foals. Two fractures were repaired with multiple intramedullary pins and the foals were euthanatized within 2 weeks due to surgical failure and, in one case, contralateral limb breakdown. The third fracture was repaired with a compressing screw and plate device; the animal was pasture sound at month 20.

Bacterial cell wall-induced immunosuppression. Role of transforming growth factor beta.

Group A streptococcal cell wall (SCW)-injected rats exhibit a profound immunosuppression that persists for months after the initial intraperitoneal injection of SCW. The goal of this study was to determine the mechanisms for the suppressed T lymphocyte proliferative responses in this experimental model of chronic inflammation. When spleen cell preparations were depleted of adherent cells, restoration of T cell proliferative responses to Con A and PHA occurred, implicating adherent macrophages in the regulation of immunosuppression. Furthermore, macrophages from SCW-treated animals, when cocultured with normal spleen cells in the presence of Con A or PHA, effectively inhibited the proliferative response. Supernatants from suppressed spleen cell cultures were found to inhibit normal T cell mitogenesis. Taken together, these results implicated a soluble macrophage-derived suppressor factor in the down regulation of T cell proliferation after exposure to SCW in vivo. Subsequent in vitro studies to identify this suppressor molecule(s) revealed the activity to be indistinguishable from the polypeptide transforming growth factor beta (TGF-beta). Furthermore, TGF-beta was identified by immunolocalization within the spleens of SCW-injected animals. The cells within the spleen that stained positively for TGF-beta were phagocytic cells that had ingested, and were presumably activated by, the SCW. These studies document that TGF-beta, previously shown to be a potent immunosuppressive agent in vitro, also effectively inhibits immune function in chronic inflammatory lesions in vivo.

Transforming growth factor-beta is a potent immunosuppressive agent that inhibits IL-1-dependent lymphocyte proliferation.

Transforming growth factor-beta (TGF-beta), a product of neoplastic and hemopoietic cells, is a bifunctional regulator of the immune response. At femtomolar concentrations, TGF-beta stimulates monocyte migration, and picomolar quantities induce synthesis of monocyte growth factors, including IL-1, that may promote tissue repair by regulating fibrosis and angiogenesis. Paradoxically, TGF-beta at picomolar concentrations also blocks the ability of IL-1 to stimulate lymphocyte proliferation. At 0.01 to 1.0 ng/ml, TGF-beta 1 and its homologue, TGF-beta 2, suppress the IL-1-dependent murine thymocyte proliferation assay. TGF-beta also inhibits human peripheral blood T lymphocyte mitogenesis. Inhibition of cell division appears to occur after activation of the lymphocytes inasmuch as neither gene expression nor translation of IL-2R is suppressed. Furthermore, TGF-beta does not block synthesis of IL-2. Therefore, TGF-beta 1 and TGF-beta 2 likely act at a site distal to IL-1 to block lymphocyte DNA synthesis. These findings suggest that TGF-beta secreted in an inflammatory site may be beneficial in diminishing lymphocyte function while promoting fibrosis and tissue repair. However, TGF-beta generated by neoplastic tissues may provide a mechanism for unrestricted tumor cell growth through its selective immunosuppressive effects.

Transforming growth factor type beta induces monocyte chemotaxis and growth factor production.

Recent studies have focused on the potential role of transforming growth factor type beta (TGF-beta) as an immunoregulatory peptide. In this context, we demonstrate that TGF-beta is a potent chemoattractant for human peripheral blood monocytes. At concentrations from 0.1 to 10 pg/ml, TGF-beta induces directed monocyte migration in vitro. Consistent with this observation is the expression of high-affinity TGF-beta receptors on the monocytes with a Kd of 1-10 pM. At higher concentrations of TGF-beta (greater than or equal to 1 ng/ml), monocytes are stimulated to generate biologically active mediator(s) that enhance fibroblast growth. Gene expression for one of these growth factors, interleukin 1, is induced in monocytes within hours after exposure to TGF-beta. Thus, TGF-beta may provide an important signal for monocyte recruitment and for regulation of their synthesis of mediators of fibroblast growth and activity in wound healing.

Monocyte interleukin 2 receptor gene expression and interleukin 2 augmentation of microbicidal activity.

Activation of human peripheral blood monocytes results in the expression of interleukin 2 (IL 2) receptors, which are absent on resting monocytes. In a population of purified monocytes, the appearance of IL 2 receptors occurs on the majority of cells in association with increased levels of HLA-DR. Lipopolysaccharide (LPS) induces maximum numbers of IL 2 receptors within 12 hr, whereas IFN-gamma requires 48 hr. We used cDNA encoding for the human IL 2 receptor to evaluate IL 2 receptor gene expression in resting and activated monocytes. Within 4 hr after LPS stimulation, IL 2 receptor mRNA species of 3500 and 1500 bases appear, reaching peak levels between 8 and 12 hr and declining thereafter. The LPS-activated monocyte IL 2 receptor protein is expressed on the cell surface within a few hours after the detection of IL 2 receptor mRNA. The addition of IL 2 to IL 2 receptor-positive monocytes augments their generation of reactive oxygen intermediates and their cytotoxic activity. Thus monocytes when activated undergo a series of morphologic, phenotypic, and functional changes, including the expression of IL 2 receptors, which may provide an important immunoregulatory pathway.

Direct toxic effect of the radiocontrast agent diatrizoate on renal proximal tubule cells.

The pathophysiology of radiocontrast agent-induced acute renal failure is presently unclear. To test for a possible direct deleterious effect of diatrizoate, a commonly used radiocontrast agent, on renal tubule cells, suspensions enriched in rabbit proximal tubule segments were incubated with sodium diatrizoate. After these manipulations, a variety of well-established metabolic parameters to quantitate the extent of cell injury were measured. Diatrizoate sodium (25 mM) produced significant declines in tubule K+, ATP, and total adenine nucleotide (TAN) contents, significant decreases in tubule basal and uncoupled respiratory rates, and a significant increase in tubule Ca2+ content, demonstrating the development of cell injury induced by diatrizoate. These effects were dose related and were progressive with increasing incubation time from 97.5 to 157.5 min. The effects of N-methylglucosamine (meglumine) on renal tubule cell viability was also evaluated. Meglumine is a low molecular weight amino-substituted cationic compound and is commonly added to radiocontrast dye solutions. Meglumine (25 mM) had significant effects to lower tubule K+ content and to decrease both tubule basal and uncoupled respiratory rates. These alterations were slightly additive to diatrizoate in that meglumine diatrizoate produced greater alterations in tubule-metabolic parameters compared to diatrizoate sodium. A period of 22.5 min of hypoxia also caused deleterious changes in each of these quantitative indices of cell viability, and diatrizoate potentiated the degree of hypoxia-induced cell injury. These results demonstrate that the radiocontrast agent, diatrizoate, is directly toxic to renal proximal tubule cells. Meglumine, a cation added to diatrizoate containing radiocontrast solutions, also had a moderate toxic effect on renal epithelial cells and added to the toxicity of diatrizoate. Diatrizoate also aggravated the degree of cell injury induced by a 22.5-min period of hypoxia. These experiments thus provide evidence for a direct toxic effect of diatrizoate on proximal renal tubule cells which was additive to hypoxic cell injury.

Bacterial cell wall-induced hepatic granulomas. An in vivo model of T cell-dependent fibrosis.

In vitro studies implicate a molecular link between inflammatory mononuclear cells and alterations in fibroblast growth and function. We have extended these observations in an experimental animal model in which we document the T cell-dependence of fibrosis that occurs after activation of the cell-mediated immune system by specific antigen. Chronic granulomatous lesions were induced in the livers of susceptible rats by the intraperitoneal injection of group A streptococcal cell walls (SCW). The development of granulomas that are composed primarily of lymphocytes and macrophages was associated with the recruitment and proliferation of connective tissue cells. Furthermore, this expanded population of fibroblasts generated a collagenous structure consisting primarily of types I and III collagen around the granuloma. The progression of these chronic inflammatory lesions leads to the formation of fibrotic nodules throughout the livers of the treated animals. Intact granulomas, as well as mononuclear cells derived from the granulomas, spontaneously elaborated a soluble factor(s) that stimulates fibroblast proliferation. Physicochemical analysis revealed that the primary granuloma-derived peak of fibroblast growth activity corresponded to an apparent Mr of 40,000, which is consistent with a previously described T lymphocyte--derived fibroblast-activating factor (FAF) in guinea pig and human. Furthermore, the fibrosis that occurs in the granuloma is apparently T cell--dependent, since no fibrotic lesions developed in SCW-injected athymic nude rats nor in SCW-injected animals treated with the T cell inhibitor, cyclosporin A (CsA). Mononuclear cells from neither of these functionally T cell--deficient animals could generate FAF activity. These data show a role for T lymphocyte--derived cytokines in the development of hepatic fibrosis in SCW-injected rats.

High energy phosphates, phospholipids, and calcium in ischemic renal tubular cell injury.

Substantial data is available supporting critical roles for high energy phosphates, calcium, and phospholipids in ischemic cell injury. Although it has been proposed that one of these compounds may be most critical and defines the "final common pathway" for lethal cell injury, it is more accurate to define the pathogenesis of ischemic cell injury as the simultaneous derangement of several critical metabolic process which act in concert to produce a cascade of events that finally lead to plasma and subcellular membrane dysfunction incompatible with the maintenance of cell viability and integrity. As detailed in Figure 1, a tentative scheme is proposed interrelating high energy phosphate depletion, cellular calcium derangements, and membrane phospholipid degradation and loss. Ischemia directly leads to declines in the rate of oxidative phosphorylation due to a lack of oxygen availability. A fall in cellular ATP levels develops. Ischemia also promotes a redistribution of intracellular calcium pools and results in phospholipase activation and phospholipid degradation. When phospholipid degradation occurs concomitantly with a decline in high energy phosphate levels, phospholipid synthesis cannot keep pace with phospholipid degradation and net membrane phospholipid loss occurs. An increase in plasma membrane calcium permeability develops and the influx of calcium down its electrochemical gradient from extracellular to intracellular spaces occurs. This calcium is taken up and sequestered in mitochondria and causes further alterations in mitochondrial structure and function, leading to further declines in cellular ATP content.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathogenetic mechanisms of nephrotoxicity: insights into cyclosporine nephrotoxicity.

Drugs may produce acute renal failure by prerenal, intrarenal and obstructive (postrenal) mechanisms. Prerenal processes usually develop from an imbalance of the normal counterbalancing vasoconstrictor and vasodilatory substances regulating RBF, resulting in a predominant vasoconstrictive state. Intrarenal processes develop from toxic renal tubule epithelial cell injury. The pathogenesis of renal cell injury is a complex interplay among derangements in subcellular membrane functions and mediators of injurious processes. Plasma and subcellular membrane injury and resulting membrane dysfunction appear most important. Cyclosporine has the ability to interact with renal tubular cell membranes in a relatively specific manner and at low concentrations. Despite this interaction, the acute declines in renal excretory function produced by cyclosporine is due predominantly to functional declines in RBF rather than structural derangements in renal tubular cell integrity. Cyclosporine-induced acute renal failure, thus, appears to be due predominantly to prerenal, rather than intrarenal, processes in the experimental animal. Cyclosporine does, however, possess a limited toxic potential to injure renal cortical cells, so that a chronic tubulointerstitial nephropathy may develop with long-term use of this immunosuppressive agent.

Cryptosporidiosis in an urban community.

Over three months Cryptosporidium oocysts were identified in faecal samples from 43 (5%) of 867 patients presenting to their general practitioners with gastrointestinal symptoms. Cryptosporidium was the second most common enteric pathogen identified. Of the 867 patients, 329 were children aged under 5, of whom 24 (7%) excreted Cryptosporidium. A characteristic clinical presentation of infection with Cryptosporidium was recognised--namely, mild gastroenteritis with four to six watery, mucoid, and offensive motions a day, which lasted for one to two weeks. The source of infection was not identified, but direct contact with farm animals was not a feature and no association with a common water supply could be established.

Comparison of carbohydrate-containing and carbohydrate-restricted hypocaloric diets in the treatment of obesity: effects of appetite and mood.

After a weight-maintaining diet base-line, obese female inpatients were provided with either a carbohydrate-restricted diet (827 kcal; 35% protein, 64% fat, 1% carbohydrate) or a carbohydrate-containing diet (827 kcal; 35% protein, 36% fat, 29% carbohydrate) for 6 wk. When compared with the psychological adjustment during the base-line diet, there was a temporary increase in appetite and a tendency toward dysphoric moods and attitudes during the 1st wk of both treatment diets. After adaptation to the treatment diets, appetite and other psychological states were similar to those during the pretreatment weight-maintaining diet. There was no support for the idea that a carbohydrate-free protein-supplemented fast decreases appetite and elevates mood in comparison with an isocaloric carbohydrate-containing diet. Thus, suppression of appetite alone does not appear to be sufficient reason in itself for using diets of this type.

Comparison of modified A-1 method with standard EC test for recovery of fecal coliform bacteria for shellfish.

This study is one of a series in which variations of the A-1 method for the detection and enumeration of fecal coliforms and Escherichia coli in seawater and foods were evaluated. The tests were conducted jointly by the Food and Drug Administration and state and provincial laboratories that support shellfish control programs in the United States and Canada as part of the National Shellfish Sanitation Program's Microbiology Task Force activities. Results showed significantly higher recovery of fecal coliforms from naturally contaminated shellfish by the AOAC official A-1-M method than by the American Public Health Association standard method. There was no significant difference in recovery of E. coli by the 2 methods.