The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.     

Comparison of sphingosine 1-phosphate-induced intracellular signaling pathways in vascular smooth muscles: differential role in vasoconstriction

Sphingosine 1-phosphate (S1P), a lipid released from activated platelets, influences physiological processes in the cardiovascular system via activation of the endothelial differentiation gene (EDG/S1P) family of 7 transmembrane G protein-coupled receptors. In cultured vascular smooth muscle (VSM) cells, S1P signaling has been shown to stimulate proliferative responses; however, its role in vasoconstriction has not been examined. In the present study, the effects of S1P and EDG/S1P receptor expression were determined in rat VSM from cerebral artery and aorta. S1P induced constriction of cerebral artery, which was partly dependent on activation of p160(ROCK) (Rho-kinase). S1P also induced activation of RhoA in cerebral artery with a similar time course to contraction. In aorta, S1P did not produce a constriction or RhoA activation. In VSM myocytes from cerebral arteries, stimulation with S1P gives rise to a global increase in [Ca2+]i, initially generated via Ca2+ release from the sarcoplasmic reticulum by an inositol 1,4,5-trisphosphate-dependent pathway. In aorta VSM, a small increase in [Ca2+]i was observed after stimulation at higher concentrations of S1P. S1P induced activation of p42/p44(mapk) in aorta and cerebral artery VSM. Subtype-specific S1P receptor antibodies revealed that the expression of S1P3/EDG-3 and S1P2/EDG-5 receptors is 4-fold higher in cerebral artery compared with aorta. S1P(1)/EDG-1 receptor expression was similar in both types of VSM. Therefore, the ability of S1P to act as a vasoactive mediator is dependent on the activation of associated signaling pathways and may vary in different VSM. This differential signaling may be related to the expression of S1P receptor subtypes.     

Smoking and disease severity in rheumatoid arthritis: Association with polymorphism at the glutathione S‐transferase M1 locus

Objective

To determine whether the relationship between smoking and disease severity in women with rheumatoid arthritis (RA) is associated with polymorphism at the glutathione S‐transferase (GST) M1 locus.

Methods

Genotyping for GSTM1 was carried out using polymerase chain reaction methodology on 164 women with established RA. Smoking history was obtained on each patient. Radiographic damage was measured by the Larsen score, and functional outcome was assessed by the Health Assessment Questionnaire (HAQ). Data were analyzed by multiple regression analyses, with correction for age and disease duration.

Results

Ever having smoked was associated with a worse radiographic and functional outcome than was never having smoked. Both past and current smoking were associated with increased disease severity. Stratification by GSTM1 status revealed that polymorphism at this locus affected the relationship between smoking and disease outcome measures. Patients who lacked the GSTM1 gene and had ever smoked had significantly higher Larsen and HAQ scores than did those who lacked the gene and had never smoked. Radiographic outcome in these patients was worse than that in patients who had the GSTM1 gene and who had smoked. The associations were not affected by correction for socioeconomic status. Rheumatoid factor (RF) production was found to be associated with smoking in only the GSTM1‐null patients.

Conclusion

Our data suggest that disease outcome in female RA patients with a history of smoking is significantly worse than in those who have never smoked. Smoking was associated with the most severe disease in patients who carried the GSTM1‐null polymorphism. This association may be due in part to a relationship between the GSTM1 polymorphism and RF production in smokers.

     

The prevalence of pain at pressure areas and pressure ulcers in hospitalised patients

Background

Patients with pressure ulcers (PUs) report that pain is their most distressing symptom, but there are few PU pain prevalence studies. We sought to estimate the prevalence of unattributed pressure area related pain (UPAR pain) which was defined as pain, soreness or discomfort reported by patients, on an “at risk” or PU skin site, reported at a patient level.

Methods

We undertook pain prevalence surveys in 2 large UK teaching hospital NHS Trusts (6 hospitals) and a district general hospital NHS Trust (3 hospitals) during their routine annual PU prevalence audits. The hospitals provide secondary and tertiary care beds in acute and elective surgery, trauma and orthopaedics, burns, medicine, elderly medicine, oncology and rehabilitation. Anonymised individual patient data were recorded by the ward nurse and PU prevalence team. The analysis of this prevalence survey included data summaries; no inferential statistical testing was planned or undertaken. Percentages were calculated using the total number of patients from the relevant population as the denominator (i.e. including all patients with missing data for that variable).

Results

A total of 3,397 patients in 9 acute hospitals were included in routine PU prevalence audits and, of these, 2010 (59.2%) patients participated in the pain prevalence study. UPAR pain prevalence was 16.3% (327/2010). 1769 patients had no PUs and of these 223 patients reported UPAR pain, a prevalence of 12.6%. Of the 241 people with pressure ulcers, 104 patients reported pain, a UPAR pain prevalence of 43.2% (104/241).

Conclusion

One in six people in acute hospitals experience UPAR pain on ‘at risk’ or PU skin sites; one in every 8 people without PUs and, more than 2 out of every five people with PUs. The results provide a clear indication that all patients should be asked if they have pain at pressure areas even when they do not have a PU.

     

Early posttranslational modifications of the three neurofilament subunits in mouse retinal ganglion cells: neuronal sites and time course in relation to subunit polymerization and axonal transport

We have characterized stages in the posttranslational processing of the three neurofilament subunits, High (NF-H), Middle (NF-M), and Low (NF-L), in retinal ganglion cells in vivo during the interval between synthesis in cell bodies within the retina and appearance of these polypeptides in axons at the level of the optic nerve (optic axons). Neurofilament proteins pulse-labeled by injecting mice intravitreally with [35S]methionine or [32P]orthophosphate, were isolated from Triton-soluble and Triton-insoluble fractions of the retina or optic axons by immunoprecipitation or immunoaffinity chromatography. Within 2 h after [35S]methionine injection, the retina contained neurofilament-immunoreactive radiolabeled proteins with apparent molecular weights of 160, 139, and 70 kDa, which co-migrated with subunits of axonal neurofilaments that were dephosphorylated in vitro with alkaline phosphatase. The two larger polypeptides were not labeled with [32P]orthophosphate, indicating that they were relatively unmodified forms of NF-H and NF-M. About 75% of the subunits were Triton-insoluble by 2 h after isotope injection, and this percentage increased to 98% by 6 h. Labeled neurofilament polypeptides appeared in optic axons as early as 2 h after injection. These subunits exhibited apparent molecular weights of 160, 139, and 70 kDa and were Triton-insoluble. The time of appearance of fully modified polypeptide forms differed for each subunit (2 h for NF-L, 6-18 h for NF-M, 18-24 h for NF-H) and was preceded by the transient appearance of intermediate forms. The modified radiolabeled subunits in optic axons 3 days after synthesis were heavily labeled with [32P]orthophosphate and exhibited the same apparent molecular weights as subunits of axonal neurofilaments (70 kDa, 145 and 140 kDa, and 195-210 kDa, respectively). Whole mounts of retina immunostained with monoclonal antibodies against NF-H in different states of phosphorylation demonstrated a transition from non-phosphorylated neurofilaments to predominantly phosphorylated ones within a region of the axon between 200 and 1000 microns downstream from the cell body. These experiments demonstrate that the addition of most phosphate groups to NF-M and NF-H takes place within a proximal region of the axon. The rapid appearance of modified forms of NF-L after synthesis may imply that processing of this subunit occurs at least partly in the cell body. The presence of a substantial pool of Triton-insoluble, unmodified subunits early after synthesis indicates that the heaviest incorporation of phosphate occurs after neurofilament proteins are polymerized.(ABSTRACT TRUNCATED AT 400 WORDS)