Chlorpromazine, quinacrine, and verapamil as donor pretreatment in liver preservation, tested in the isolated perfused rat liver.

Rats were pretreated with a single iv dose of chlorpromazine (CPZ) 3 mg/kg, verapamil 1 mg/kg, or quinacrine 2 mg/kg. Livers were taken out and perfused with University of Wisconsin (UW) preservation solution and stored on ice for 48 h in the UW solution before reperfusion with erythrocyte-free and colloid-free Krebs-Hanseleit buffer at 38 degrees C in a nonrecirculating perfusion system for 2 h. CPZ- and quinacrine-pretreated livers produced significantly more bile than control livers and also released significantly less alanine aminotransferase into the perfusate at 30, 60, and 120 min of reperfusion. Aspartate aminotransferase levels were lower at 30 and 60 min of reperfusion for CPZ-pretreated livers but not at 120 min. The only difference between groups concerning lactate dehydrogenase (LDH) release into the perfusate was that CPZ decreased the amount of LDH released at 60 min. Total tissue water or tissue electrolyte content of the liver tissue at the end of the reperfusion did not differ between groups. In conclusion, verapamil was ineffective when given as single dose iv pretreatment to the liver donor but pretreatment with CPZ or quinacrine appeared to improve the function of the preserved liver.     

Mitogenic activity of pituitary hormones on cell cultures of normal and carcinogen-induced tumor epithelium from rat mammary glands

Cell suspension containing normal or tumor epithelium were readily obtained by enzymatically digesting rat mammary glands from perphenazine-treated (prolactin-hypersecreting) cycling, female virgin animals or hormone- responsive mammary tumors from animal treated with dimethylbenzanthracene. Cell suspensions were fractioned into predominantly epithelial and predominantly stromal cells by their differential rates of attachment to culture dishes. Both normal mammary and tumor epithelial cells were characterized by the presence of specific cell-junctional complexes, desmosome-like structures, surface microvilli, and their ability to synthesize casein. Serum-dependent protease activity was greater in cultures derived from tumors, and cells from such cultures grew in agarose whereas those from the non-neoplastic gland did not. The addition of prolactin to the culture medium stimulated DNA synthesis in primary or secondary epithelial cultures from tumors, whereas additional insulin and hydrocortisone with prolactin were required for similar levels of DNA synthesis in cultures from non-neoplastic glands. The fraction of cells synthesizing DNA was, however, smaller than that with 10 percent serum measured in the same time period. Both growth hormone and epidermal growth factor stimulated DNA synthesis but to a lesser extent than did prolactin. Prolactin with hydrocortisone and insulin were relatively inactive in promoting DNA synthesis of the nonepithelial cells whereas pituitary fibroblast growth factor was more active. These mitogenic effects were obtained when the hormones were added to the medium at near physiological concentrations, and paralleled the known activities of the hormones in control of mammary gland growth and development in the rat.     

Factors influencing the anterior component of occlusal force

We hypothesized that the anterior component of occlusal force (ACF) generated by mandibular molars was a function of molar inclination, height of the transverse condylar axis above the occlusal plane, steepness of the occlusal plane, gape, molar root dimensions, interproximal tooth contact force when not biting, and bite force. Our research aim was to identify those biomechanical factors which determine ACF. Mandibular second molars were axially loaded with a 90 N force (10 mm second molar gape) in 15 subjects, and the resulting ACF was measured at the mandibular first molar-second premolar contact using a recording technique based on interproximal frictional forces. Morphologic measurements were obtained from lateral cephalometric radiographs of each subject and included: Frankfort mandibular plane angle, occlusal plane angle, angles formed by the longitudinal axis of the second molar and the occlusal and mandibular planes, perpendicular distance from the top of the condyle to the occlusal plane, and second molar root width and root length. For ten subjects, ACF resulting from axial loads of 50, 100, 150, and 200 N was measured. For ten subjects, ACF resulting from an axial load of 50 N and second molar gapes of 10 mm, 14 mm, 18 mm, and 22 mm were measured. ACF increased with increasing gape and increased proportionally to increasing bite force. Correlation and stepwise regression analyses revealed that ACF varies with interproximal tooth contact force when not biting (contact ‘tightness’) and molar root width (model R² = 0.71, p < 0.01). The hypothesis that ACF is a function of bite force, gape, molar root width, and interproximal contact tightness has been supported, and the hypothesis that ACF is a function of molar inclination, occlusal plane steepness, condylar axis height, and root length was rejected.     

On the origins of esterases

Comparisons among the primary sequences of five cloned eukaryotic esterasesreveal two distinct lineages, neither bearing any significant overallsequence similarity to the functionally related serine protease multigenefamily. We have not eliminated the possibility that the esterases may haveresidual conformational similarities to the serine proteases. However, ourprofile analysis and analyses of the predicted conformations of theesterases reveal little similarity to the serine proteases. Four of theesterase proteins share 27%-53% overall sequence similarity and evidence ofa catalytic mechanism involving the same Arg- Asp-Ser or His-Asp-Ser chargerelay. We propose that these four esterases, three of them cholinesterases,form part of a multigene family essentially separate from the serineproteases.     

Specific inhibition of phosphate transport in mitochondria by N-ethylmaleimide

N-ethylmaleimide (NEM), a reagent that alkylates free sulfhydryl groups, was shown to be a highly effective inhibitor of the following coupled mitochondrial processes: oxidative phosphorylation, ATP-³²Pi exchange, Pi-induced light scattering and configurational changes, State III respiration, valinomycin-induced translocation of potassium with Pi as the anion, and calcium accumulation in presence of Pi. However, NEM was less effective or ineffective in inhibiting some processes that do not require inorganic Pi, namely electron transfer and ATPase activity, ADP binding, energized light scattering changes induced by arsenate and nonenergized light scattering changes induced by acetate. The rate of oxidative phosphorylation and of ATP-³²Pi exchange was normal in ETP_H particles prepared from NEM-treated mitochondria. Also NEM, even et levels 2–3 times greater than those required to inhibit oxidative phosphorylation in intact mitochondria, did not inhibit coupled processes in submitochondrial particles. We are proposing that NEM alkylates sulfhydryl groups in the mitochondrion that modulate Pi translocation, and that the suppression of Pi translocation blocks oxidative phosphorylation, the Pi-dependent energized configurational change in mitochondria and Pi-dependent transport processes.On leave of absence from the Department of Biochemistry, Cancer Institute Okayama University Medical School, Okayama, Japan.On leave of absence from the Department of Pathology, Nagoya University Medical School, Nagoya, Japan.     

Viability assays in organ preservation

Assays to determine the viability of preserved organs ideally must meet two important requirements: (i) in the clinical environment, they should allow the surgeon to determine if an organ will be viable when it is transplanted (this needs to be done in a noninvasive, nondestructive manner, and currently no such assay exists), and (ii) in the research environment, they should aid in the development of improved methods of organ preservation. Currently, however, the only reliable means of assessing viability is actual transplantation. Many conventional biochemical and physiological techniques have been used to describe the mechanism of preservation-induced injury and to help improve preservation. This paper reviews some techniques that have been used to aid in the development of organ preservation.     

Preservation of metabolic reserves and function after storage of myocytes in hypothermic UW solution

Isolated rat myocytes coldstored anaerobically up to 24 h in University of Wisconsinsolution lost 95% of their ATP and 100% of their glycogen. Theyunderwent contracture when rewarmed in a Krebs-Henseleit (KH) mediumthat contained Ca unless Ca addition was delayed. In the latter case,cell function, measured by stimulation-induced cell shortening, wassurprisingly well retained. Aerobically stored cells were resistant toCa on rewarming, although 96% of glycogen was still lost, along with46% of ATP. Cells that were incubated for 48 h aerobically withthe substrates glucose and pyruvate at pH 6.2 retained 77% of theirATP and 59% of their glycogen, with good cell morphology. At pH 6.2, the demand for ATP was only 55% of that at pH 7.4. However, afterrewarming, these cells functioned no better than anaerobically storedcells, although their inotropic response to isoproterenol was improved.We conclude that 1) aerobic conditions with substrates atlow pH preserve myocyte metabolic reserves well for 48 h, partlyby reducing the demand for ATP; 2) rewarming conditions arecritical for anaerobically stored cells with metabolic stores that areseverely depleted; and 3) unloaded cell function issurprisingly insensitive to a period of severe metabolic deprivation.

     

Inflammatory thresholds and the species-specific effects of colonising bacteria in stable chronic obstructive pulmonary disease

Background

There has been increasing interest in the use of newer, culture-independent techniques to study the airway microbiome of COPD patients. We investigated the relationships between the three common potentially pathogenic microorganisms (PPMs) Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis, as detected by quantitative PCR (qPCR), and inflammation and health status in stable patients in the London COPD cohort.

Methods

We prospectively collected sputum, serum and plasma samples for analysis of airway bacterial presence and load, and airway and systemic inflammation from 99 stable COPD patients between January 2011 and October 2012. Health status was measured with St George’s Respiratory Questionnaire and COPD Assessment Test.

Results

Airway inflammation and plasma fibrinogen, but not C-reactive protein, were greater in samples with PPM detection (p < 0.001, p = 0.049 and p = 0.261, respectively). Increasing total bacterial load was associated with increasing airway (p < 0.01) but not systemic inflammation (p > 0.05). Samples with high total bacterial loads had significantly higher airway inflammation than both samples without PPM detection and those with lower loads. Haemophilus influenzae presence was associated with significantly higher levels of airway but not systemic inflammation for all given pathogen loads (p < 0.05), and was significantly greater than with other PPMs. No association was observed between inflammation and health status (p > 0.05).

Conclusions

Airway and systemic inflammation, as measured by fibrinogen, is greater in stable COPD patients with PPMs detected using the culture-independent qPCR technique. The airway, but not systemic inflammatory response, appears to have a total pathogen-load threshold and appears attributable to Haemophilus influenzae. However, discordance between inflammation and health status was observed.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0114-1) contains supplementary material, which is available to authorized users.