Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain.

Pleckstrin homology (PH) domains are found in many signaling molecules and are thought to be involved in specific intermolecular interactions. Their binding to several proteins and to membranes containing 1-alpha-phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been reported. A region that includes the PH domain has also been implicated in binding of phospholipase C-delta 1 (PLC-delta 1) to both PtdIns(4,5)P2 and D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] [Cifuentes, M. E., Delaney, T. & Rebecchi, M. J. (1994) J. Biol. Chem. 269, 1945-1948]. We report herein that the isolated PH domain from PLC-delta 1 binds to both PtdIns(4,5)P2 and Ins(1,4,5)P3 with high affinity and shows the same binding specificity seen by others with whole PLC-delta 1. Thus the PH domain is functionally and structurally modular. These results demonstrate stereo-specific high-affinity binding by an isolated PH domain and further support a functional role for PH domains in the regulation of PLC isoforms. Other PH domains did not bind strongly to the compounds tested, suggesting that inositol phosphates and phospholipids are not likely physiological ligands for all PH domains. Nonetheless, since all PH-domain-containing proteins are associated with membrane surfaces, several PH domains bind to specific sites on membranes, and PH domains appear to be electrostatically polarized, a possible general role for PH domains in membrane association is suggested.     

Molecular phylogenetic inference from saber-toothed cat fossils of Rancho La Brea.

A method for the successful extraction of sequestered cellular DNA from 14,000-year-old fossil bones was developed and applied to asphalt-preserved specimens of the extinct saber-toothed cat, Smilodon fatalis. Two distinct gene segments, the mitochondrial gene for 12S rRNA and nuclear FLA-I (the feline class I major histocompatibility complex gene), from three different individual fossil specimens were cloned and sequenced after PCR amplification. Comparison of fossil-derived DNA sequences to homologous regions in 15 living carnivorous species, including 9 species of Felidae and 6 nonfelids, affirmed the phylogenetic placement of Smilodon within the modern radiation of Felidae distinct from the Miocene paleofelid (Nimravidae) saber-toothed "cat" species. These results raise the prospect of obtaining genetically informative DNA from preserved bones of extinct fossil species, particularly among the 2 million specimens excavated from the asphaltic sediments at Rancho La Brea in metropolitan Los Angeles.     

The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains.

rho-like GTP binding proteins play an essential role in regulating cell growth and actin polymerization. These molecular switches are positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP. Using the interaction-trap assay to identify candidate proteins that bind the cytoplasmic region of the LAR transmembrane protein tyrosine phosphatase (PT-Pase), we isolated a cDNA encoding a 2861-amino acid protein termed Trio that contains three enzyme domains: two functional GEF domains and a protein serine/threonine kinase (PSK) domain. One of the Trio GEF domains (Trio GEF-D1) has rac-specific GEF activity, while the other Trio GEF domain (Trio GEF-D2) has rho-specific activity. The C-terminal PSK domain is adjacent to an Ig-like domain and is most similar to calcium/calmodulin-dependent kinases, such as smooth muscle myosin light chain kinase which similarly contains associated Ig-like domains. Near the N terminus, Trio has four spectrin-like repeats that may play a role in intracellular targeting. Northern blot analysis indicates that Trio has a broad tissue distribution. Trio appears to be phosphorylated only on serine residues, suggesting that Trio is not a LAR substrate, but rather that it forms a complex with LAR. As the LAR PTPase localizes to the ends of focal adhesions, we propose that LAR and the Trio GEF/PSK may orchestrate cell-matrix and cytoskeletal rearrangements necessary for cell migration.     

Effects of saliva contamination and phosphoric acid composition on bond strength

The purpose of this study was to determine the effects of a phosphoric acid gel and solution, and saliva contamination of etched enamel on the tensile bond strength (TBS) of a posterior composite resin to etched enamel. Ninety-six extracted human permanent maxillary canines were used in this study. The test system developed by Kemper and Kilian was used to determine the TBS of a posterior composite resin (P-10) to enamel. The ground enamel surfaces were etched with 37% H₃PO₄ gel for 60 sec and washed for 15 sec or 30 sec, and with 37% H₃PO₄ solution for 60 sec and washed for 15 sec or 30 sec, respectively. The ground enamel surfaces were also etched with H₃PO₄ gel for 60 sec, washed for 15 sec and rewashed for 15 sec after saliva contamination of the etched enamel for 15 sec, 60 sec and 60 min, respectively. Etched enamel surfaces contaminated with saliva for 15 sec were re-etched for 15 sec and washed for 15 sec. The test specimens were mounted in an Instron machine and subjected to a tensile load at 0.02 inch. min⁻¹. The force required to break a test specimen was recorded, and the bond strengths were calculated and expressed in MN.m⁻². The data were analyzed by ANOVA and Duncan's multiple range test at the 5% level of significance. The H₃PO₄ composition (gel vs solution) and wash times had no significantly different effect on TBS. Only a 60 min saliva contamination significantly reduced TBS. In this study the TBS was determined 24 h after specimen preparation. It is possible that salivary contamination of etched enamel may have an adverse effect on bond strength only after prolonged immersion of the test specimens prior to testing.     

Persistent superior oblique paresis as a manifestation of familial periodic cerebellar ataxia.

A brother and sister complained of persistent diplopia due to superior oblique palsies. The cause of their symptoms became apparent when they were diagnosed as having familial periodic cerebellar ataxia (FPCA), a rare autosomal dominant condition. Oral acetazolamide (250 mg twice daily) not only prevented all the periodic symptoms but also relieved their diplopia, which had been present between attacks.     

EEG dipole source localisation of interictal spikes acquired during routine clinical video-EEG monitoring

OBJECTIVE: We investigated the feasibility of electroencephalography (EEG) dipole source localisation of interictal epileptiform discharges from data acquired during routine clinical inpatient video-EEG monitoring (VEM) and compared a 19-channel 'routine montage' with a 29-channel 'surgical montage' that includes an additional row of 10 inferior temporal electrodes. METHODS: Twenty consecutive patients who had VEM for the presurgical evaluation of medically refractory partial epilepsy were screened. Thirteen of the patients had focal interictal spikes recorded, and in 11 (85%) these were technically satisfactory for source localisation. Fourteen spike foci were analysed as 3 patients had bilateral independent spikes. EEG data was acquired with 29 electrodes including an inferior temporal row (surgical montage). For comparison, the 10 additional electrodes were excluded from analysis (routine montage). Using NEUROSCAN Source 2.0 software, a computed dipole source localisation of averaged spikes was performed utilising a magnetic resonance imaging-based finite element model. Dipole localisation was compared with that of the Comprehensive Epilepsy Program (CEP) evaluation. RESULTS: Using the surgical montage dipole source localisation was consistent with the CEP spike localisation for 13/14 spikes (93%, P<0.005), compared with only 5/14 spikes (36%) using the routine montage. CONCLUSIONS: Data derived from routine clinical inpatient VEM using a routine montage can yield accurate EEG dipole source localisation, but significantly more accurate localisation is obtained using the surgical montage.     

Relation between severity of Alzheimer''s disease and costs of caring

BACKGROUND: Data from the Canadian Study of Health and Aging (CSHA) were used to examine the relation between severity of Alzheimer's disease, as measured by the Mini-Mental State Examination (MMSE), and costs of caring. METHODS: The CSHA was a community-based survey of the prevalence of dementia, including subtypes such as Alzheimer's disease, among elderly Canadians. Survey subjects with a diagnosis of possible or probable Alzheimer's disease were grouped into disease severity levels of mild (MMSE score 21-26), mild to moderate (MMSE score 15-20), moderate (MMSE score 10-14) and severe (MMSE score below 10). Components of care available from the CSHA were use of nursing home care, use of medications, use of community support services by caregivers and unpaid caregiver time. Costs were calculated from a societal perspective and are expressed in 1996 Canadian dollars. RESULTS: The annual societal cost of care per patient increased significantly with severity of Alzheimer's disease. The cost per patient was estimated to be $9451 for mild disease, $16,054 for mild to moderate disease, $25,724 for moderate disease and $36,794 for severe disease. Institutionalization was the largest component of cost, accounting for as much as 84% of the cost for people with severe disease. For subjects living in the community, unpaid caregiver time and use of community services were the greatest components of cost and increased with disease severity. INTERPRETATION: The societal cost of care of Alzheimer's disease increases drastically with increasing disease severity. Institutionalization is responsible for the largest cost component.     

Antihypertensive and renal haemodynamic effects of atenolol and nadolol in elderly hypertensive patients.

As little is known of the antihypertensive efficacy or renal haemodynamic effects of beta-adrenoceptor blocking drugs in the elderly we studied two such drugs, atenolol and nadolol, in elderly hypertensive patients. Ten patients took part in a placebo-controlled double-blind study of atenolol and 10 received nadolol in a single-blind placebo-controlled study. Treatment phases lasted 12 weeks for atenolol or 10 weeks for nadolol. Blood pressure, effective renal blood flow and glomerular filtration rate data obtained at the end of each treatment phase were analysed. Atenolol lowered mean arterial pressure (mean +/- s.e. mean) from 129.9 +/- 1.5 to 108.2 +/- 2.3 mm Hg (P less than 0.01) while it increased mean effective renal blood flow 512.5 +/- 86.6 to 646.0 +/- 116.1 ml min-1 1.73 m-2 (P less than 0.05). Nadolol reduced mean arterial pressure from 133.2 +/- 2.0 to 113.5 +/- 3 mm Hg (P less than 0.001) but reduced mean effective renal blood flow from 558.8 +/- 32.2 to 446.0 +/- 26.9 ml min-1 1.73 m-2 (P less than 0.05). Glomerular filtration did not alter significantly with either drug. We conclude that beta-adrenoceptor blocking drugs are effective antihypertensive agents in the elderly but have disparate effects on effective renal blood flow perhaps because of differences in cardioselectivity. These data suggest that comparative studies with thiazide diuretics and beta-adrenoceptor blocking drugs are warranted in elderly hypertensives.     

A pharmacokinetic/pharmacodynamic/receptor binding model to predict the onset and duration of pharmacological activity of the benzodiazepines

1. 1. Ex vivo receptor binding as a function of time was determined in Charles River rats.

2. 2. The pharmacokinetic and protein binding parameters in man as well as the ex vivo receptor binding parameters in rat brain for three benzodiazepine induction agents, diazepam, lorazepam and midazolam, were used to develop and test a pharmacokinetic/ pharmacodynamic/receptor binding model.

3. 3. The model was subsequently used to predict changes in receptor binding and pharmaco-dynamics as a function of changes in pharmacokinetics.

4. 4. The model was found to be a good predictor of the relative onset and duration of the sedative and amnesic properties in normal subjects as well as in the presence of certain patho-physiological conditions and certain drug interactions.

Osteoid osteoma: excision with scintimetric guidance

The definite diagnosis of osteoid osteoma relies on the demonstration of the nidus, best shown by CT, which also provides precise preoperative localization of the nidus. While bone-block excision to remove the nidus is feasible in the long bones, there may be unacceptable sequelae in the vertebral column and small bones of the hands. By precisely localizing the nidus, radionuclide scintimetry permits excellent therapeutic results, with minimal morbidity.