Genetic divergence and evolutionary instability in ospE-related members of the upstream homology box gene family in Borrelia burgdorferi sensu lato complex isolates

A series of related genes that are flanked at their 5' ends by a conserved upstream sequence element called the upstream homology box (UHB) have been identified in Borrelia burgdorferi. These genes have been referred to as the UHB or erp gene family. We previously demonstrated that among a limited number of B. burgdorferi isolates, the UHB gene family is variable in composition and organization. Prior to this report the UHB gene family in other species of the B. burgdorferi sensu lato complex had not been studied, and if this family is important in the pathogenesis or biology of the Lyme disease spirochetes, then a wide distribution among species and isolates of the B. burgdorferi sensu lato complex would be expected. To assess this, we screened for the UHB element by Southern hybridization and determined its restriction fragment length polymorphism (RFLP) patterns. The UHB element was found to be carried by all B. burgdorferi sensu lato complex species tested (B. burgdorferi, B. garinii, B. afzelii, B. japonica, B. valaisiana sp. nov., and B. andersonii), but the RFLP patterns varied widely at both the inter- and intraspecies levels. Variation in both the number and size of the hybridizing restriction fragments was evident. PCR analyses also revealed the presence of polymorphic, ospE-related alleles in many isolates. Sequence analyses identified the molecular basis of the polymorphisms as being primarily insertions and deletions. Sequence variation and the insertions and deletions were found to be clustered in two distinct domains (variable domains 1 and 2). In many isolates variable domain 1 is flanked by direct repeat elements, some as long as 38 bp. Computer analyses of the deduced amino acid sequences encoded within variable domain 1 predict them to be hydrophilic, surface exposed, and antigenic. The analyses conducted here suggest that the UHB gene family, as evidenced by the variable UHB RFLP patterns, is not evolutionarily stable and that the polymorphic ospE alleles are derived from a common ancestral gene which has been modified through mutation or recombination events. The characterization of ospE-related genes of the UHB gene family among B. burgdorferi sensu lato species will prove important in attempts to construct a model for UHB gene family organization and in deciphering the role of the UHB gene family in the biology and pathogenesis of the Lyme disease spirochetes.     

Langerhans' cell histiocytosis of the spine: use of MRI in guiding biopsy

The MRI features of two cases of spinal Langerhans' cell histiocytosis with multilevel involvement are presented in which MRI was of help in differentiating active from inactive healing lesions by the demonstration of signal changes in the vertebral body marrow of the active lesion, manifest as low signal intensity on T1-weighted sequences and high signal intensity on T2-weighted sequences. This distinction could not be made by plain radiography or bone scintigraphy. In cases where biopsy is required for diagnosis, MRI is recommended to guide the biopsy towards levels suggestive of active involvement.     

Root stimulation studies in the evaluation of patients with motor neuron disease

Nerve root stimulation may be employed in patients with motor neuron disease (MND) to rule out motor neuropathy with conduction block. The diagnostic utility of these studies is unknown, in part because the range of amplitude changes across nerve root segments in patients with active neuronal degeneration has not been well studied. We reviewed root stimulation studies in 32 patients (59 nerves) with MND and found segmental amplitude reduction from 0 to 45%, a range similar to values reported for normal subjects; there was no suggestion of conduction block based on our usual criteria.     

The CRY1 blue light photoreceptor of Arabidopsis interacts with phytochrome A in vitro

Plants have at least two major photosensory receptors: phytochrome (absorbing primarily red/far-red light) and cryptochrome (absorbing blue/UV-A light); considerable physiological and genetic evidence suggests some form of communication or functional dependence between the receptors. Here, we demonstrate in vitro, using purified recombinant photoreceptors, that Arabidopsis CRY1 and CRY2 (cryptochrome) are substrates for phosphorylation by a phytochrome A-associated kinase activity. Several mutations within the CRY1 C terminus lead to reduced phosphorylation by phytochrome preparations in vitro. Yeast two-hybrid interaction studies using expressed C-terminal fragments of CRY1 and phytochrome A from Arabidopsis confirm a direct physical interaction between both photoreceptors. In vivo labeling studies and specific mutant alleles of CRY1, which interfere with the function of phytochrome, suggest the possible relevance of these findings in vivo.     

Familial neuroblastoma: report of a kindred with later age at diagnosis

The first reported effective adjuvant combination regimen for patients with operable breast cancer comprised oral cyclophosphamide (C) days 1-14 with intravenous methotrexate (M) and fluorouracil (F) on days 1 and 8, repeated every 28 days ('classical' CMF). These drugs have since been extensively used with or without endocrine therapies and/or other cytotoxics, as well as with radiation therapy to the chest wall yielding conflicting results. Although doses and schedules have varied widely, the combination of these three drugs has been generically referred to as CMF. Evidence exists that reducing the dose and/or altering the schedule of CMF ('modified' CMF) have compromised its efficacy in metastatic breast cancer. Reduction below standard dose of a similar regimen also gave inferior results in the adjuvant setting. In fact, the recently reported improved outcome of adding radiation therapy to CMF was only demonstrated in comparisons with a 'modified' CMF. Furthermore, trials in women with estrogen receptor-positive breast cancer, which did not demonstrate any significant benefit for the addition of adjuvant CMF to tamoxifen compared with tamoxifen alone, also used 'modified' CMF. Therefore, adherence to the 'classical' dose and schedule is recommended when CMF is used in adjuvant therapy.     

State-dependent NMDA receptor antagonism by Ro 8-4304, a novel NR2B selective, non-competitive, voltage-independent antagonist

1. Subunit-selective blockade of N-methyl-D-aspartate (NMDA) receptors provides a potentially attractive strategy for neuroprotection in the absence of undesirable side effects. Here, we describe a novel NR2B-selective NMDA antagonist, 4-?3-[4-(4-fluoro-phenyl)-3,6-dihydro-2H-pyridin-1-yl]-2-hydroxy-propoxy ?-benzamide (Ro 8-4304), which exhibits >100 fold higher affinity for recombinant NR1(001)/NR2B than NR1(001)/NR2A receptors. 2. Ro 8-4304 is a voltage-independent, non-competitive antagonist of NMDA receptors in rat cultured cortical neurones and exhibits a state-dependent mode of action similar to that described for ifenprodil. 3. The apparent affinity of Ro 8-4304 for the NMDA receptor increased in an NMDA concentration-dependent manner so that Ro 8-4304 inhibited 10 and 100 microM NMDA responses with IC50s of 2.3 and 0.36 microM, respectively. Currents elicited by 1 microM NMDA were slightly potentiated in the presence of 10 microM Ro 8-4304, and Ro 8-4304 binding slowed the rate of glutamate dissociation from NMDA receptors. 4. These results were predicted by a reaction scheme in which Ro 8-4304 exhibits a 14 and 23 fold higher affinity for the activated and desensitized states of the NMDA receptor, respectively, relative to the agonist-unbound resting state. Additionally, Ro 8-4304 binding resulted in a 3 4 fold increase in receptor affinity for glutamate site agonists. 5. Surprisingly, whilst exhibiting a similar affinity for NR2B-containing NMDA receptors as ifenprodil, Ro 8-4304 exhibited markedly faster kinetics of binding and unbinding to the NMDA receptor. This spectrum of kinetic behaviour reveals a further important feature of this emerging class of NR2B-selective compounds.     

Tuberculosis 2000: problems and solutions

The incidence of tuberculosis is expected to increase, from 8.8 million cases in 1995, to 10.2 million cases by the year 2000 and 11.9 million by 2005. Three million deaths due to tuberculosis occurred in 1995, and 3.5 million can be expected in the year 2000. The most important causes of the world-wide increase in tuberculosis are: 1) non-compliance with control programmes; 2) inadequate diagnosis and treatment; 3) migration; 4) endemic human immunodeficiency virus (HIV); 5) ambulatory and self-administered treatment. In the 1970s it was stated that treatment needed to be supervised-a recommendation that went unheeded. A number of fundamental changes should be introduced in order to make treatment effective, to cure patients and thus to arrest the transmission of the disease: 1) supervision during the whole period antituberculosis drugs are taken, and 2) hospitalization during the initial treatment stage for all groups at risk. It is already 50 years since the first antituberculosis drugs were discovered; effective treatments capable of curing all patients in 6 months have been available for the last 25 years, and the result is failure plus a growing mortality curve at the beginning of the twenty-first century. If we wish to alter this trend, we need trained doctors all over the world who possess enough clinical knowledge of tuberculosis; hospitalization for specific groups of tuberculosis patients; true supervision during the whole treatment period; fixed-dose combinations of drugs; and prophylaxis or preventive treatment whenever possible. We also need to take into account other factors such as drug resistance, endemic HIV, and migration.     

Negative regulation of Wee1 expression and Cdc2 phosphorylation during p53-mediated growth arrest and apoptosis

The G2 cell cycle checkpoint protects cells from potentially lethal mitotic entry after DNA damage. This checkpoint involves inhibitory phosphorylation of Cdc2 at the tyrosine-15 (Y15) position, mediated in part by the Wee1 protein kinase. Recent evidence suggests that p53 may accelerate mitotic entry after DNA damage and that the override of the G2 checkpoint may play a role in the induction of apoptosis by p53. To determine the biochemical mechanism by which p53 inactivates the G2 checkpoint, the effects of p53 activation on Wee1 expression, Cdc2-Y15 phosphorylation, and cyclin B1-associated Cdc2 kinase activity were examined. Under conditions of either growth arrest or apoptosis, p53 activation resulted in the down-regulation of Wee1 expression and dephosphorylation of Cdc2. A parallel increase in cyclin B1/Cdc2 kinase activity was observed during p53-mediated apoptosis. Negative regulation of the Wee1 expression and Cdc2 phosphorylation by p53 was also evident in thymus tissue from p53+/+ mice but not from p53-/- mice. Inactivation of the G2 checkpoint may contribute to the tumor suppressor activity of p53.