Characterizing replication intermediates in the amplified CHO dihydrofolate reductase domain by two novel gel electrophoretic techniques

Using neutral/neutral and neutral/alkaline two-dimensional (2-D) gel techniques, we previously obtained evidence that initiation can occur at any of a large number of sites distributed throughout a broad initiation zone in the dihydrofolate reductase (DHFR) domain of Chinese hamster ovary (CHO) cells. However, other techniques have suggested a much more circumscribed mode of initiation in this locus. This dichotomy has raised the issue whether the patterns of replicating DNA on 2-D gels have been misinterpreted and, in some cases, may represent such noncanonical replication intermediates as broken bubbles or microbubbles. In an accompanying study (R. F. Kalejta and J. L. Hamlin, Mol. Cell. Biol. 16:4915-4922, 1996), we have shown that broken bubbles migrate to unique positions in three different gel systems and therefore are not likely to be confused with classic replication intermediates. Here, we have applied a broken bubble assay developed from that study to an analysis of the amplified DHFR locus in CHO cells. This assay gives information about the number and positions of initiation sites within a fragment. In addition, we have analyzed the DHFR locus by a novel stop-and-go-alkaline gel technique that measures the size of nascent strands at all positions along each arc in a neutral/neutral 2-D gel. Results of these analyses support the view that the 2-D gel patterns previously assigned to classic, intact replication bubbles and single-forked structures indeed correspond to these entities. Furthermore, potential nascent-strand start sites appear to be distributed at very frequent intervals along the template in the intergenic region in the DHFR domain.     

Morphological and functional polymorphism within clonal thyroid nodules

Thirty-nine thyroid nodules, removed because of recent growth, were analyzed morphologically by serial histological sections for the classical histomorphological hallmarks of follicular cell replication and for immunohistochemically demonstrable overexpression of the growth-associated ras-gene product p21ras. Clonal analysis was performed using the highly informative probe M27 beta that detects polymorphisms on the locus DXS255 of the X-chromosome. Twenty-four nodules were of clonal and 15 nodules were of poly-clonal origin. Only 3 out of the 24 clonal nodules were histomorphologically uniform. In all others, the structural hallmarks of active growth and the P21ras growth-marker expression were remarkably heterogeneous throughout the tumors. There were no histomorphological characteristics distinguishing these clonal tumors from polyclonal nodules. Even if a clonal thyroid tumor may be originally homogeneous in respect to the parameters studied here, mechanisms must exist that create wide heterogeneity of growth and of morphogenetic potential among the individual follicular cells during further expansion of the nodule. Thus, clonal nodules are much more common in nodular goiters than hitherto assumed on grounds of the classical morphological criteria. The diagnosis of a true monoclonal nodule can no longer rely on morphological and functional criteria alone but requires molecular or cytogenetic analysis of clonality.     

The miswak chewing stick: a traditional oral hygiene aid

The miswak chewing stick is an oral hygiene device used by the majority of people in Arab Gulf countries. Despite its widespread use, few studies demonstrated its benefits or applications as an alternative and convenient means for cleansing the teeth. This paper will examine the unique properties of the miswak chewing stick and its proper use.     

Design flaw can convert commercially available continuous syringe pumps to intermittent bolus injectors

OBJECTIVE: To investigate if unexpected behaviour of neonatal and paediatric patients connected to syringe pumps could be explained by transient elevation of these devices. DESIGN: Five different commercially available syringe-pumps were set at an infusion rate of 1 ml/h and then subjected to a vertical displacement manoeuvre (height 1 m). The actual delivered infusion volumes in association with the displacement manoeuvre were measured by a high precision weight scale connected to a computer. SETTING: A medical technology laboratory in a university hospital. MEASUREMENTS AND RESULTS: Elevation of the devices resulted in a rapid bolus injection of 0.19-2.28 ml. Returning the devices to their original positions resulted in an aspiration into the system of 0.06-0.34 ml. The times both for bolus injection and for aspiration into the system were less than 1 min in all cases. The updown manoeuvre was followed by a period with zero infusion ranging from 8 to 105 min. CONCLUSIONS: Design flaws in the construction of syringe pumps can expose patients to substantial danger following vertical displacement if potent drugs are being infused. If potent drugs are infused, care should be taken not to change the vertical position of the syringe pump even for short periods of time. Before buying new equipment, the authors recommend that the delivery characteristics of these devices should not only be tested during ordinary bench testing but should also include the reaction to a vertical displacement manoeuvre.     

Seasonal variation in the role of grey squirrels as hosts of Ixodes ricinus, the tick vector of the Lyme disease spirochaete, in a British woodland

The partition of free fatty acids (FFA) to egg-phosphatidylcholine (egg-PC) and egg-phosphatidylethanolamine (egg-PE) vesicles was studied. Upon the addition of FFA to the suspension of vesicles, the pH of the aqueous phase changed depending on the length and saturation of the FFA hydrocarbon chain, as well as on the vesicle composition. The medium pH decreased faster if FFA was added to egg-PE as compared to egg-PC vesicles. The fluorescent free fatty acid indicator (ADIFAB) was used to measure the amount of FFA remaining in the aqueous phase. Most of the FFA added to the suspension of egg-PE vesicles remained in the aqueous phase, whereas in the presence of egg-PC vesicles the FFA partitioned preferentially into the lipid phase. The amount of FFA incorporated into the lipid bilayers was estimated by measuring the changes of pH at the lipid bilayer surface, using fluorescein-PE. At high surface concentrations of FFA, decreasing pH at the bilayer surface caused the protonation of FFA, and raised the pK of FFA at the bilayer surface from 5 to about 7. The partition of FFA in egg-PE vesicles was an order of magnitude lower than that in egg-PC vesicles. The incorporation amount was determined more by the molecular packing than by the nature of lipid headgroups, because steroylcaprioyl-PE, which preferred the bilayer structure, behaved more like egg-PC than egg-PE. Understanding FFA partition characteristics would help to interpret the hydrolysis measurements of phospholipids, and to explain many biological activities of FFA.