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.     

StAR protein is expressed in both medulla and cortex of the bovine and rat adrenal gland

We have employed polyclonal antibodies to a peptide sequence of bovine steroidogenic acute regulatory (StAR) protein and human placental 3beta-hydroxysteroid dehydrogenase (3beta-HSD) to determine the localisation and distribution of these proteins in rat and bovine adrenal glands. Immunohistochemical staining demonstrated the presence of StAR protein in the zona glomerulosa (ZG), zona fasciculata (ZF), zona reticularis (ZR) and in the medulla of both species. For 3beta-HSD, immunostaining was observed in the ZG, ZF and ZR of the rat adrenal and was absent in the medulla. Immunoblotting experiments showed intense bands for StAR protein (30 kDa, 37 kDa) in the mitochondria of bovine ZG, ZF and medulla and a less intense band (30 kDa) in the microsomes. In rat ZG and ZF/R mitochondria only the 30 kDa protein was present. For 3beta-HSD, an intense band (42 kDa) was found in microsomes and mitochondria of rat and bovine ZG and ZFR. A very faint signal for 3beta-HSD was seen in adrenal medulla. In conclusion, StAR (or a closely related) protein is present throughout the adrenal gland in rat and bovine species in contrast to 3beta-HSD which is confined to the steroidogenic zones. The possible function of StAR protein in the adrenal medulla merits investigation.     

A case-mix classification system for medical rehabilitation

Dissatisfaction with Medicare's current system of paying for rehabilitation care has led to proposals for a rehabilitation prospective payment system, but first a classification system for rehabilitation patients must be created. Data for 36,980 patients admitted to and discharged from 125 rehabilitation facilities between January 1, 1990, and April 19, 1991, were provided by the Uniform Data System for Medical Rehabilitation. Classification rules were formed using clinical judgment and a recursive partitioning algorithm. The Functional Independence Measure version of the Function Related Groups (FIM-FRGs) uses four predictor variables: diagnosis leading to disability, admission scores for motor and cognitive functional status subscales as measured by the Functional Independence Measure, and patient age. The system contains 53 FRGs and explains 31.3% of the variance in the natural logarithm length of stay for patients in a validation sample. The FIM-FRG classification system is conceptually simple and stable when tested on a validation sample. The classification system contains a manageable number of groups, and may represent a solution to the problem of classifying medical rehabilitation patients for payment, facility planning, and research on the outcomes, quality, and cost of rehabilitation.     

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.     

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.     

The histologic anatomy of the volar plate

The EU Concerted Action Workshop on 11q23 Abnormalities in Hematological Malignancies collected 550 patients with abnormalities involving 11q23. Of these, 53 patients had a translocation involving chromosome 11, breakpoint q23, and chromosome 19, breakpoint p13. Karyogram review enabled each patient to be further defined as t(11;19)(q23;p13.1) (21 patients) or t(11;19)(q23;p13.3) (32 patients). There was a marked difference between the type of banding and the translocation identified: t(11;19)(q23;p13.1) was detected predominantly by R-banding, whereas t(11;19)(q23;p13.3) was detected almost solely by G-banding. Additional change was extremely rare in patients with t(11;19)(q23;p13.1) but occurred in nearly half of the patients with t(11;19)(q23;p13.3). Patients with t(11;19)(q23;p13.1) all had leukemia of a myeloid lineage, mostly acute myeloid leukemia (AML), and were predominantly adult. In contrast patients with t(11;19)(q23;p13.3) had malignancies of both myeloid and lymphoid lineage and were mainly infants less than 1 year old. The survival of both groups of patients was generally poor, over 50% of t(11;19)(q23;p13.1) patients died within 2 years of diagnosis and the median survival of acute lymphoblastic leukemia (ALL) patients with t(11;19)(q23;p13.3) was 17.6 months.