A sensitive analytical method for the detection and quantitation of adenosine in biological samples

A new method for the measurement of adenosine in biological materials has been developed. The method is based on the combined principles of isotope dilution and enzymatic catalysis using a highly specific adenosine kinase isolated from rat heart. By differential centrifugation and gel filtration, this adenosine kinase was obtained free of adenosine deaminase and other enzymes which would have been a source of error in the use of this enzyme in the adenosine assay. The cardiac adenosine kinase was shown to be highly specific and to exhibit an apparent K_m for adenosine of 0.35 μM. Using this enzyme, unknown quantities of adenosine could be detected by measuring the effect of their addition on the conversion of radioisotopic adenosine to 5′-AMP in the kinase reaction. In this procedure, as little as 20 pmoles of adenosine could be detected. To test the applicability of the assay, measurements of the tissue content of this nucleoside were made in samples of dog and rat hearts frozen in situ under control, hypoxic, or ischemic conditions. The assay has several advantageous features when compared to other existing methods used to measure adenosine: a minimum of sample preparation is required before the actual assay procedure; many samples can be processed per day by a single operator; single determinations can be done on as little as 5 μl of sample, and the specificity of the assay can be readily checked by treatment of samples with adenosine deaminase.     

The phosphorylation of ribosomal protein S6 in baby hamster kidney fibroblasts

Ribosomal protein S6 was extensively phosphorylated in pre-confluent but not in post-confluent baby hamster kidney fibroblasts. This appears to be the first example of increased phosphorylation of S6 under physiological conditions where the cellular concentration of cyclic AMP is not elevated. The extent of the phosphorylation of S6 was also independent of alterations in the protein synthetic activity of the cells, suggesting that the biological role of this phosphorylation may be unrelated to the functional ability of the ribosomes.     

The variable location of phosphoproteins on eukaryotic ribosomal subunits

A pair of phosphoproteins of low molecular weight detected on the 60S subunit of ribosomes isolated from Krebs II ascites cells are on the 40S subunit of ribosomes isolated from BHK-21/C13 cells. The possibility that these proteins may be located at the interface between the two subunits is discussed.     

Therapeutic donor insemination with frozen semen.

Although it is now accepted that cryopreserved semen must, on ethical and medicolegal grounds, be used for donor insemination many clinicians still believe that it has an unacceptably reduced fecundability rate as compared with fresh semen. We studied the outcome of 81 recipients who started therapeutic donor insemination (TDI) treatment during 1986 in a program that used exclusively cryopreserved semen; 55 had never undergone TDI and were receiving the first series (six cycles), 6 were receiving the second series (also six cycles), and 20 had achieved pregnancy through TDI previously and were starting the treatment again. Insemination with semen stored in 0.5-ml French straws was performed daily during the periovulatory period while the modified Insler score was 10 or greater out of 15. A total of 42 (52%) of the recipients became pregnant within six TDI cycles; 4 (10%) had a spontaneous abortion. An average of 4.8 straws were used per cycle among those who became pregnant and 5.1 per cycle among those who did not. On average 2.6 cycles were required to achieve pregnancy. The overall fecundability rate was 14.6%. We conclude that a TDI program involving exclusively frozen semen can be operated with a success rate comparable to rates achieved with fresh semen if a simple, established cryopreservation method and an uncomplicated clinical management protocol are used.     

Effects of anions on cellular volume and transepithelial Na+ transport across toad urinary bladder

Summary The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl^– on transepithelial Na⁺ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (V _T) decreased rapidly, transepithelial resistance (R _T) increased, and calculated short-circuit current (I _sc) decreased. CalculatedE _Na was unaffected, indicating that the inhibition of Na⁺ transport was a consequence of a decreased apical membrane Na⁺ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateV _T decreased,R _T increased andI _sc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedE _Na fell substantially (from 130 to 68 mV on average). Ba²⁺ (3mm) reduced calculatedE _Na in Cl^– Ringer's but not in gluconate Ringer's. With replacement of serosal Cl^– by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andE _Na did not fall. Replacement of serosal isosmotic Cl^– medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na⁺ transport. Thus the inhibition of Na⁺ transport can be correlated with changes in cell volume rather than with the change in Cl^– per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K⁺ gluconate mucosa, Na⁺ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na⁺/K⁺ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl^– results in a loss of highly specific Ba²⁺-sensitive K⁺ conductance channels from the basolateral plasma membrane. It is possible that the number of Na⁺ pump sites in this membrane is also decreased.     

Pulmonary Fissure Integrity and Collateral Ventilation in COPD Patients

Purpose

To investigate whether the integrity (completeness) of pulmonary fissures affects pulmonary function in patients with chronic obstructive pulmonary disease (COPD).

Materials and Methods

A dataset consisting of 573 CT exams acquired on different subjects was collected from a COPD study. According to the global initiative for chronic obstructive lung disease (GOLD) criteria, these subjects (examinations) were classified into five different subgroups, namely non-COPD (222 subjects), GOLD-I (83 subjects), GOLD-II (141 subjects), GOLD-III (63 subjects), and GOLD-IV (64 subjects), in terms of disease severity. An available computer tool was used to aid in an objective and efficient quantification of fissure integrity. The correlations between fissure integrity, and pulmonary functions (e.g., FEV1, and FEV1/FVC) and COPD severity were assessed using Pearson and Spearman''s correlation coefficients, respectively.

Results

For the five sub-groups ranging from non-COPD to GOLD-IV, the average integrities of the right oblique fissure (ROF) were 81.8%, 82.4%, 81.8%, 82.8%, and 80.2%, respectively; the average integrities of the right horizontal fissure (RHF) were 62.6%, 61.8%, 62.1%, 62.2%, and 62.3%, respectively; the average integrities of the left oblique fissure (LOF) were 82.0%, 83.2%, 81.7%, 82.0%, and 78.4%, respectively; and the average integrities of all fissures in the entire lung were 78.0%, 78.6%, 78.1%, 78.5%, and 76.4%, respectively. Their Pearson correlation coefficients with FEV1 and FE1/FVC range from 0.027 to 0.248 with p values larger than 0.05. Their Spearman correlation coefficients with COPD severity except GOLD-IV range from −0.013 to −0.073 with p values larger than 0.08.

Conclusion

There is no significant difference in fissure integrity for patients with different levels of disease severity, suggesting that the development of COPD does not change the completeness of pulmonary fissures and incomplete fissures alone may not contribute to the collateral ventilation.     

The use of fresh embryos in stem cell research: ethical and policy issues

Fresh embryos resulting from in vitro fertilization, including many of poor quality, can provide sources of human embryonic stem cell lines. We consider why some donate such embryos for this research, address relevant ethical and policy issues, and present core guidelines for fresh embryo donation based on those of Canada.