Nutrient Restriction Increases Circulating and Hepatic Ceramide in Dairy Cows Displaying Impaired Insulin Tolerance.

The progression of insulin resistance in dairy cows represents a maternal adaptation to support milk production during heightened energy demand; however, excessive adipose tissue lipolysis can develop. In diabetic non-ruminants, the mechanisms that mediate insulin resistance involve the sphingolipid ceramide. We tested the hypothesis that ceramide accumulates in dairy cows experiencing lipolysis and insulin resistance. Nine dairy cows were utilized in a replicated 3 × 3 Latin square design. Cows were ad libitum fed, nutrient-restricted (NR), or NR with nicotinic acid (NA; 5 mg of NA/h per kg BW; delivered i.v.) for 34 h. When provided access, cows were ad libitum fed a mixed ration of grass hay and ground corn to meet requirements. Intake for NR cows was limited to vitamins and minerals. Nicotinic acid was administered to suppress lipolysis. Saline was infused in cows not provided NA. At 32 and 33 h of treatment, a liver biopsy and insulin tolerance test were performed, respectively. Samples were analyzed using colorimetry, immunoassay, and mass spectrometry. Nutrient restriction increased serum fatty acids and ceramide levels, and impaired insulin sensitivity; however, NA infusion was unable to prevent these responses. We also show that NR increases hepatic ceramide accumulation, a response that was positively associated with serum ceramide supply. Our data demonstrate that circulating and hepatic 24:0-Cer are inversely associated with systemic insulin tolerance, an effect not observed for the 16:0 moiety. In conclusion, our results suggest that ceramide accrual represents a metabolic adaptation to nutrient restriction and impaired insulin action in dairy cows.

Regulation of eicosanoid synthesis in microvessel endothelium: glucocorticoids do not affect arachidonyl CoA synthetase activity.

The properties of acyl coenzyme A (CoA) synthetase activity were characterized in cultured rabbit coronary microvessel endothelial cells. We report here that microvessel endothelial cells contain two long-chain acyl CoA synthetases. One shows activity with a variety of fatty acids, similar to long-chain non-selective fatty acyl CoA synthetases described previously. The other activity was selective for arachidonic acid and other structurally related substrates. Both activities required ATP, Mg2+ and CoA for optimal activity. The arachidonyl CoA and the non-selective acyl CoA synthetases showed different thermolabilities. Arachidonyl CoA formation was inhibited by greater than 50% after 1 min at 45 degrees C, whereas a 15 min heating treatment was necessary to produce the same relative inhibition of oleoyl CoA synthesis. Glucocorticoid pretreatment (10(-7) M dexamethasone) of the RCME cells did not affect the apparent Km or Vmax, nor the fatty acid selectivity for either acyl CoA synthetase. Therefore, although fatty acyl CoA synthetases may be involved in limiting eicosanoid formation, these activities do not appear to be glucocorticoid-responsive.

Arachidonic acid metabolism by cultured bovine corneal endothelial cells.

Pathways of arachidonic acid metabolism were identified in freshly prepared and in cultured bovine corneal endothelial cells. The principal pathway of arachidonic acid metabolism in the bovine corneal endothelial cells appears to be the cyclooxygenase pathway with the resultant synthesis of PGI2, PGF2 alpha and PGE2. At least two of these products, PGI2 and PGF2 alpha, are formed by the enzymatic conversion of the substrate, PGH2. Measurements of endogenous prostaglandin production by radioimmunoassay demonstrated that PGE2 was the major arachidonic acid metabolite released, with smaller amounts of PGF2 alpha and the stable hydrolysis product of PGI2, 6-keto PGF1 alpha. The release of all three prostanoids was significantly increased by the addition of the calcium ionophore (A23187), human thrombin, bradykinin and histamine. Basal and stimulated release of prostaglandins by the corneal endothelium may contribute to the regulation of intraocular pressure and also in the modulation of the corneal response to injury.

Proteoglycans in the human sclera. Evidence for the presence of aggrecan.

PURPOSE: The proteoglycans synthesized and accumulated within the adult human sclera (aged 50 to 80 years) were identified by their size, glycosaminoglycan side chains, and core proteins in an effort to characterize the proteoglycan content of the human sclera. METHODS: Sclerae, unlabeled, or radiolabeled in organ culture with 35SO4 or 3H-proline, were extracted in 4M guanidine-HCl and separated by Sepharose CL-2B and Superose 6 forced-pressure liquid chromatography. Peak fractions, identified by glycosaminoglycan content or radioactivity, were pooled and subjected to G-50 chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis before and after digestion with specific glycosidases. Scleral proteoglycan core proteins were identified in Western blot analysis using specific antisera to decorin, biglycan, and aggrecan. Reverse transcription-polymerase chain reaction analyses were carried out on human scleral fibroblast RNA to confirm the transcription of one scleral proteoglycan. Proteoglycans were localized on sections of scleral tissue using specific antisera. RESULTS: After chromatography on CL-2B, scleral proteoglycans could be resolved into three major peaks, PG-1, PG-2, and PG-3. The largest scleral proteoglycan, PG-1, contained chondroitin sulfate and keratan sulfate glycosaminoglycan side chains. Results of Western blot analyses indicated that the core protein of PG-1 is the aggrecan core protein, migrating at approximately 350 kDa. Reverse transcription-polymerase chain reaction analyses confirmed that human scleral fibroblasts transcribe aggrecan in vitro and in vivo. PG-2 and PG-3 were identified as biglycan and decorin in Western blot analyses using antibiglycan and antidecorin antibodies, respectively. Immunostaining results indicated that aggrecan, biglycan, and decorin are distributed throughout the thickness of the human sclera. CONCLUSIONS: The adult human sclera contains three major proteoglycans; aggrecan, biglycan, and decorin. It is likely that these proteoglycans contribute to the structural properties of the sclera and that the ratios of these proteoglycans will change with age, specific region, and condition of the sclera.

Gelatinase A and TIMP-2 expression in the fibrous sclera of myopic and recovering chick eyes.

PURPOSE: Myopia, or nearsightedness, is characterized by excessive lengthening of the ocular globe and is associated with extracellular matrix remodeling in the posterior sclera. The activity of gelatinase A, a member of the matrix metalloproteinase family, has been shown to increase in the posterior sclera during the development of induced myopia in several species. In the present study, the distribution and relative expression of gelatinase A and its associated inhibitor, tissue inhibitor of metalloproteinases (TIMP)-2, were measured within the fibrous scleras of experimentally myopic (form-deprived) eyes, control eyes, and eyes recovering from form deprivation to better understand the mechanisms that regulate scleral remodeling and the rate of ocular elongation. METHODS: Total RNA was extracted from the posterior scleras of form-deprived chick eyes, eyes recovering from deprivation myopia, and paired contralateral control eyes, and subjected to northern blot analysis analyses using cDNA probes to chicken gelatinase A and TIMP-2. The distribution of gelatinase A and TIMP-2 mRNAs was evaluated by in situ hybridization on frozen sections of chick scleras using 33P-labeled RNA probes. Gelatinase A activity within the fibrous scleras of form-deprived eyes and paired contralateral recovering eyes was evaluated by gelatin zymography. RESULTS: Northern blot analysis indicated that the relative expression of gelatinase A was increased by 128% in deprived eyes (P = 0.009), whereas after 1 day of recovery, levels were decreased by 80% in scleras from recovering eyes (P = 0.005). In contrast, TIMP-2 expression was significantly decreased (-53%, P = 0.027) in the posterior scleras of form-deprived eyes. No significant differences were detected in levels of TIMP-2 expression between recovering eyes and paired control eyes. In situ hybridization indicated that most of the gelatinase A transcripts were present in the fibrous layer of the posterior scleras from form-deprived and recovering eyes. CONCLUSIONS: Changes in the steady state levels of gelatinase A and TIMP-2 mRNA lead to changes in gelatinase activity within the fibrous sclera and mediate, at least in part, the process of visually regulated ocular growth and scleral remodeling.

Alteration of platelet aggregation by cigarette smoke and carbon monoxide.

Platelet aggregation with epinephrine, adenosine diphosphate and arachidonic acid was studied in the presence of cigarette smoke and carbon monoxide. It is shown that cigarette smoke inhibits the arachidonic acid induced platelet aggregation as well as the second phase of epinephrine induced aggregation. The adenosine diphosphate induced platelet aggregation is not significantly affected by cigarette smoke. Carbon monoxide causes similar alterations in platelet aggregation. These results suggest that cigarette smoke inhibits platelet aggregation. This aggregation inhibition is due to the presence of carbon monoxide.

The ability of fibrinogens, FI and FII, to support ADP-induced platelet aggregation.

Fibrinogen plays an integral part in ADP-induced platelet aggregation. Controversy exists in regard to the role of the carboxy termini of fibrinogen A alpha chains in this reaction. We have attempted to clarify this problem in view of the availability of a highly purified FII fibrinogen fraction. Kabi fibrinogen or its purified fractions FI, FII and FIII-IV-V were added to washed platelets in the presence of Tyrode-HEPES buffer pH 7.4. Aggregation was initiated by the addition of calcium and ADP. These fibrinogen fractions equally promoted ADP-induced platelet aggregation. The major difference among these fractions is in their A alpha chains. The FI fraction contains intact A alpha chains while FII and FIII-IV-V fractions have one and two partially degraded A alpha chains at the carboxy terminal portion respectively. We conclude that the carboxy terminal portion of the A alpha chain does not play an important role in promoting ADP-induced platelet aggregation.

Health effects of salicylates in foods and drugs.

There is much (renewed) interest about the effects of salicylates on food intolerance, attention-deficit disorders, and cardiovascular disease. Current evidence for the efficacy of salicylate-elimination diets in the treatment of attention-deficit disorders and hyperactivity is weak, and further investigation is required on the relationship between salicylates and cardiovascular disease.

Analysis of potential risks associated with 7.5% sodium chloride resuscitation of traumatic shock.

We evaluated the potential side effects of rapidly infusing 250 mL of either 7.5% sodium chloride or 7.5% sodium chloride per 6% dextran 70, using lactated Ringer's as the control, to 106 critically injured patients in two prospective double-blinded emergency department trials. Eight patients had a significant hyperchloremic acidemia in association with infusion of the hypertonic solutions, but all eight were moribund before infusion and many factors other than hyperchloremia could have contributed to their acidemia. Other blood chemistry changes that might have been associated with the hypertonic solutions, such as hyperosmolality or hypernatremia, were made insignificant by other factors, such as high blood alcohol levels or concomitant administration of sodium bicarbonate. There were no cases of central pontine myelinolysis; bleeding was not potentiated. There was no difficulty with crossmatching of blood. No anaphylactoid reactions occurred. In a setting of limited volume resuscitation, the solutions are likely to have a favorable risk-to-benefit ratio.

Early fluid requirements in trauma patients. A predictor of pulmonary failure and mortality.

The fluid required for initial resuscitation of trauma patients should reflect, at least in part, the severity of the original injuries and shock. We examined the hypothesis that the initial fluid requirements might also predict development of subsequent pulmonary failure and death. Fluid balances were calculated for the first 24 hours in the intensive care unit for 100 high-risk trauma patients. The mean (+/- 1 SD) fluid balance for 63 patients who developed pulmonary failure was 4.6 +/- 5.5 L; the mean balances for the 37 patients who did not develop pulmonary failure were 1.0 +/- 3.1 L. The balances in 23 patients who died and in 77 who survived were 6.8 +/- 5.4 and 2.2 +/- 4.5 L, respectively. A cutoff value of 3 L determined prospectively before beginning the study predicted pulmonary failure with a sensitivity of 52% and a specificity of 89%. For mortality, the 3-L cutoff point gave a sensitivity of 74% and a specificity of 74%. The predictive value of the fluid balance was independent of other prognostic indicators, such as revised trauma scores, injury Severity Scores, and modified APACHE II scores. This simple measurement should help in allocating intensive care unit resources, as patients in positive fluid balance are likely to require Swan-Ganz catheterization and are likely to require long-term mechanical ventilation. The fluid balance should also be useful in stratifying patients for entry into clinical trials.

7.5% sodium chloride/dextran for resuscitation of trauma patients undergoing helicopter transport.

To evaluate the use of hypertonic saline/dextran solutions in the prehospital resuscitation of severely injured patients, we administered 250 mL of either 7.5% sodium chloride/dextran 70 (HSD) (n = 83) or lactated Ringer's solution (n = 83), followed by conventional isotonic fluids, to 166 trauma patients with systolic blood pressures less than or equal to 100 mm Hg, in a prospective, randomized, double-blinded clinical trial. Patients in the sodium chloride/dextran 70 group required less fluid before hospitalization and arrived in the emergency department with higher systolic blood pressures than patients in the lactated Ringer's solution group. The rate of survival to hospital discharge for the entire cohort was 64% for patients in the sodium chloride/dextran 70 group vs 59% for patients in the lactated Ringer's solution group. The rate of survival to hospital discharge for the patients with severe head injuries was 32% for the sodium chloride/dextran 70 group vs 16% for the lactated Ringer's solution group. Actuarial survival for patients with severe head injuries in the sodium chloride/dextran 70 group compared with patients with severe head injuries in the lactated Ringer's solution group did not quite reach statistical significance. There were no adverse side effects associated with sodium chloride/dextran 70 administration. Administration of small volumes of sodium chloride/dextran 70 before hospitalization increased the blood pressure of severely injured patients more effectively than did lactated Ringer's solution and showed tendencies toward improving survival in the patients with severe head injuries.

Evaluation of prostaglandin E1 for prevention of respiratory failure in high risk trauma patients: a prospective clinical trial and correlation with plasma suppressive factors for neutrophil activation.

A group of 48 critically injured patients were entered into a prospective, double-blind, placebo-controlled trial to evaluate the efficacy of early infusion of PGE1 for reducing the incidence of severe respiratory failure and mortality. Secondary assessments examined the effects of the PGE1 infusion on plasma mediated suppression of PMN superoxide production and loss of PMN granule enzyme content. The incidence of severe respiratory failure was lower in the PGE1 group--13% versus 32%, but this did not reach significance. The overall morality was equivalent between the two groups--26% (PGE1) versus 28% (placebo). The suppressive activity of the patient plasma was assayed by measurement of normal PMN superoxide production relative to normal control plasma (ratio P:C). The baseline ratio P:C was 62 +/- 5% in the PGE1 group versus 60 +/- 5% in the placebo group. The day 1 plasma samples showed significant reversal of plasma suppressive activity in the PGE1 group--ratio P:C 88 +/- 5% versus 67 +/- 5% in the placebo group (P less than 0.02). In patients who received the full 7 days of infusion, the plasma suppressive activity remained significantly diminished in the PGE1 group--ratio P:C 77 +/- 4% versus 61 +/- 5% (P less than 0.04). The baseline lysozyme content of patient PMN's relative to that of normal control PMNs (ratio P:C) was 119 +/- 14% in the PGE1 group. A significant loss of lysozyme content was observed in the PGE1 group on day 1 of the infusion--ratio P:C 79 +/- 8% (P less than 0.03), and was associated with a reduction in the plasma suppressive activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Perspectives on clinical trials for hypertonic saline/dextran solutions for the treatment of traumatic shock.

Animal studies with hypertonic solutions suggest that they can achieve resuscitation of hypovolemic shock with extremely small volumes. Such small volume resuscitation might be ideal in the field treatment of injured patients. Our studies to date, with 60 patients entered into a prospective, randomized, placebo-controlled, and double-blind clinical trial, suggest that the use of a 7.5% NaCl/Dextran 70 solution increases blood pressures during transport. The solutions have been safe, and we have encountered no adverse side effects from their use. Survival rates to date favor use of the solutions, but we do not have convincing statistical significance yet in that regard.

The snRNP E protein multigene family contains five pseudogenes with common mutations.

Sequence data from three previously-uncharacterized members of the snRNP E protein multigene family suggest that each is a non-transcribed processed pseudogene, even though one clone has the potential to code for a full-length protein with greater than 90% similarity to previously-characterized E protein cDNAs. Each of the newly-analyzed family members is without introns, contains a tract of polyadenylic acid residues, and is flanked by short direct repeats. In addition, the three sequences all contain point mutations that distinguish them from the E protein coding sequence. Seven point mutations are common to the three sequences described here and to two previously-described E protein pseudogenes. Although all of these mutations are transitions, only 5 of 7 could have been generated by deamination of methylated cytosines in inactive genes. Thus, the common mutations in the pseudogenes suggest an origin other than the expressed gene that we have described. Allelic variants for two of the pseudogenes were detected and repetitive elements are located near four of the five E protein pseudogenes that have been characterized.

Knowledge bases in medicine: a review.

Efforts to represent knowledge effectively have been central to progress in various aspects of medical informatics. These efforts range from relatively simple "electronic textbooks" to fairly sophisticated knowledge-based systems, which function as well as, or even better than, human experts faced with similar problems. Knowledge bases have been developed in many fields, but the relatively limited domains and structured language of medicine, as well as the importance of information in the provision of good medical care, have made research in medical knowledge representation an area of intense activity. This paper reviews representative knowledge bases and knowledge-based systems in medicine: electronic textbooks such as PDQ and the Hepatitis Knowledge Base (HKB), rule-based systems such as MYCIN, causal models (e.g., CASNET), and hypothesis- or frame-based systems, exemplified by PIP and INTERNIST-1. The paper describes the relationships among divergent approaches and provides a sense of current and future trends. It examines problems in knowledge-based systems, particularly in knowledge representation and acquisition, and the responses to these challenges. The latter include the use of domain-independent software shells for constructing knowledge bases, the adaptation and use of previously existing knowledge bases, and multiple uses of the same knowledge base for different purposes.

Online information retrieval in pharmacy and related fields.

Online information retrieval in pharmacy and related fields is described. Factors involved in determining whether to conduct an online search are discussed, including characteristics of appropriate and less suitable topics, advantages and limitations of online searching versus manual searching, and possible types of searches. The process of preparing for an online search, involving the determination of search vocabulary, relevant citations, important authors, time frame, special categories (such as language, publication type, and reviews), and the number of citations needed, as well as choosing a database, is explained. Sample search strategies on MEDLINE and IPA are illustrated to demonstrate the basic search commands and to compare file retrievals on the sample subject. Pharmacy-related bibliographic databases, general-interest databases, end-user search services, and full-text and numeric databases are profiled. Online database searching can be a cost-efficient and flexible alternative to manual literature searching for pharmacists. Although most online searching is currently conducted by librarian-search specialists, end-user searching is a growing trend, as is the availability of full-text databases.

Hemoglobin autoxidation at physiological concentrations.

Methemoglobin formation was studied at near physiological hemoglobin concentration. The reaction proceeds at a faster rate when the concentration of hemoglobin is high (15-18 mM in heme) than when it is low (2 mM). Constant shaking of hemoglobin preparations during the incubation decreases the differences seen in the rates of autoxidation between concentrated and dilute samples. When red cell hemolysate is used instead of pure hemoglobin, similar results are obtained. A comparison of rates of methemoglobin formation in hemoglobin solutions under low air pressure (1/2 atm) with those under normal air pressure (1 atm) shows no differences between concentrated and dilute samples. There is also no significant difference between the rates of autoxidation of dilute and concentrated solutions when the reactions are carried out under one atmosphere of oxygen (100 percent O2). The study of one patient with hereditary spherocytosis demonstrated higher hemoglobin autoxidation rate in spherocytes, which have higher hemoglobin concentration, than in normal biconcave red cells. These results suggest that: a) the rate of hemoglobin autoxidation at red cell hemoglobin concentration is significantly faster than rates obtained by studying dilute solutions; b) although the accelerated oxidation might be related to multiple factors, one seems to be less accessibility of oxygen when the hemoglobin solution is highly concentrated.

Influence of histone acetylation on the solubility, H1 content and DNase I sensitivity of newly assembled chromatin.

In a previous report [Annunziato, A.T. and Seale, R.L. (1983) J. Biol. Chem. 258:12675] a novel intermediate in chromatin assembly was described (detected by labeling new DNA in the presence of the deacetylase inhibitor sodium butyrate), which retained approximately 50% of the heightened sensitivity of newly replicated chromatin to DNaseI. It is now reported that nucleosomes replicated in butyrate are considerably more soluble in the presence of magnesium, relative to chromatin replicated under control conditions, and that this heightened magnesium-solubility is reflected in a concomitant increase in the preferential solubility of nucleosomes containing newly synthesized core histones. This differential solubility was accompanied by a 5- to 6-fold depletion of histone H1, and was completely abolished by the selective removal of H1 from isolated nuclei. The removal of H1 also markedly reduced the preferential DNaseI sensitivity of chromatin replicated in butyrate. Further, when mononucleosomes of control and (acetylated) nascent chromatin were compared, no differences in DNaseI sensitivity were detected. These results provide evidence that the interactions between newly assembled nucleosomes and histone H1 are altered when histone deacetylation is inhibited during chromatin replication, and suggest a mechanism for the control of H1 deposition during nucleosome assembly in vivo.

Histone acetylation reduces H1-mediated nucleosome interactions during chromatin assembly.

During chromatin replication and nucleosome assembly, newly synthesized histone H4 is acetylated before it is deposited onto DNA, then deacetylated as assembly proceeds. In a previous study (Perry and Annunziato, Nucleic Acids Res. 17, 4275 [1989]) it was shown that when replication occurs in the presence of sodium butyrate (thereby inhibiting histone deacetylation), nascent chromatin fails to mature fully and instead remains preferentially sensitive to DNaseI, more soluble in magnesium, and depleted of histone H1 (relative to mature chromatin). In the following report the relationships between chromatin replication, histone acetylation, and H1-mediated nucleosome aggregation were further investigated. Chromatin was replicated in the presence or absence of sodium butyrate; isolated nucleosomes were stripped of linker histone, reconstituted with H1, and treated to produce Mg(2+)-soluble and Mg(2+)-insoluble chromatin fractions. Following the removal of H1, all solubility differences between chromatin replicated in sodium butyrate for 30 min (bu-chromatin) and control chromatin were lost. Reconstitution with H1 completely restored the preferential Mg(2+)-solubility of bu-chromatin, demonstrating that a reduced capacity for aggregation/condensation is an inherent feature of acetylated nascent nucleosomes; however, titration with excess H1 caused the solubility differences to be lost again. Moreover, when the core histone N-terminal "tails" (the sites of acetylation) were removed by trypsinization prior to reconstitution, H1 was unable to reestablish the altered solubility of chromatin replicated in butyrate. Thus, the core histone "tails," and the acetylation thereof, not only modulate H1-mediated nucleosome interactions in vitro, but also strongly influence the ability of H1 to differentiate between new and old nucleosomes. The data suggest a possible mechanism for the control of H1 deposition and/or chromatin folding during nucleosome assembly.

Geophysical, archaeological, and historical evidence support a solar-output model for climate change.

Although the processes of climate change are not completely understood, an important causal candidate is variation in total solar output. Reported cycles in various climate-proxy data show a tendency to emulate a fundamental harmonic sequence of a basic solar-cycle length (11 years) multiplied by 2(N) (where N equals a positive or negative integer). A simple additive model for total solar-output variations was developed by superimposing a progression of fundamental harmonic cycles with slightly increasing amplitudes. The timeline of the model was calibrated to the Pleistocene/Holocene boundary at 9,000 years before present. The calibrated model was compared with geophysical, archaeological, and historical evidence of warm or cold climates during the Holocene. The evidence of periods of several centuries of cooler climates worldwide called "little ice ages," similar to the period anno Domini (A.D.) 1280-1860 and reoccurring approximately every 1,300 years, corresponds well with fluctuations in modeled solar output. A more detailed examination of the climate sensitive history of the last 1, 000 years further supports the model. Extrapolation of the model into the future suggests a gradual cooling during the next few centuries with intermittent minor warmups and a return to near little-ice-age conditions within the next 500 years. This cool period then may be followed approximately 1,500 years from now by a return to altithermal conditions similar to the previous Holocene Maximum.