Tumefactive cyst with a vascular blush as a late complication after combined embolization and stereotactic radiosurgery treatments for a cerebral arteriovenous malformation.

Cyst formation is a recognized late complication after stereotactic radiosurgery for cerebral arteriovenous malformations (AVMs). We report on a patient with delayed cyst formation after combined embolization and stereotactic radiosurgery treatments for a cerebral AVM. The true nature of the cyst was complicated by tumefactive magnetic resonance MR imaging characteristics. The tumefactive cyst was associated with an additional imaging finding suggestive of a neoplastic lesion - a 'blush' on conventional angiography.

Acetate non-utilizing mutants of Arabidopsis: evidence that organic acids influence carbohydrate perception in germinating seedlings.

A phenotypic screen was employed to isolate Arabidopsis plants that are deficient in their ability to utilize or sense acetate. The screening strategy, based on resistance to the toxic acetate analogue monofluoroacetic acid, was adapted from one that has been used successfully to identify important metabolic and regulatory genes involved in acetate metabolism in fungi. Following conventions established from the fungal work, the mutants were called acn mutants for acetate non-utilization. Three highly resistant plant lines were the focus of genetic and physiological studies. Mutant acn1 appears to be a true acetate non-utilizing mutant, as it displays increased sensitivity to exogenous acetate. The progeny of the original acn2 mutant did not germinate, even in the presence of sucrose as an exogenous carbon source. The germination of seeds from the F3 generation depended on the sucrose concentration in the medium. Only a small proportion of seeds germinated in the absence of exogenous sucrose and in the presence of 100 mM sucrose, but up to 70% of seeds germinated on 20 mM sucrose. Mutant acn3 exhibited sensitivity to exogenous sucrose, showing significant chlorosis on medium containing 20 mM sucrose, but no chlorosis when grown in the absence of exogenous sucrose. This phenotype was alleviated if acetate was provided. The acn mutants demonstrate that disrupting organic acid utilization can have profound affects on carbohydrate metabolism.

Structural basis for SRY-dependent 46-X,Y sex reversal: modulation of DNA bending by a naturally occurring point mutation.

The HMG-box domain of the human male sex-determining factor SRY, hSRY(HMG) (comprising residues 57-140 of the full-length sequence), binds DNA sequence-specifically in the minor groove, resulting in substantial DNA bending. The majority of point mutations resulting in 46X,Y sex reversal are located within this domain. One clinical de novo mutation, M64I in the full-length hSRY sequence, which corresponds to M9I in the present hSRY(HMG) construct, acts principally by reducing the extent of DNA bending. To elucidate the structural consequences of the M9I mutation, we have solved the 3D solution structures of wild-type and M9I hSRY(HMG) complexed to a DNA 14mer by NMR, including the use of residual dipolar couplings to derive long-range orientational information. We show that the average bend angle (derived from an ensemble of 400 simulated annealing structures for each complex) is reduced by approximately 13 degrees from 54(+/-2) degrees in the wild-type complex to 41(+/-2) degrees in the M9I complex. The difference in DNA bending can be localized directly to changes in roll and tilt angles in the ApA base-pair step involved in interactions with residue 9 and partial intercalation of Ile13. The larger bend angle in the wild-type complex arises as a direct consequence of steric repulsion of the sugar of the second adenine by the bulky S(delta) atom of Met9, whose position is fixed by a hydrogen bond with the guanidino group of Arg17. In the M9I mutant, this hydrogen bond can no longer occur, and the less bulky C(gamma)m methyl group of Ile9 braces the sugar moieties of the two adenine residues, thereby decreasing the roll and tilt angles at the ApA step by approximately 8 degrees and approximately 5 degrees, respectively, and resulting in an overall difference in bend angle of approximately 13 degrees between the two complexes. To our knowledge, this is one of the first examples where the effects of a clinical mutation involving a protein-DNA complex have been visualized at the atomic level.

31P NMR analysis of the DNA conformation induced by protein binding SRY/DNA complexes.

Complexes of the HMG box protein SRY with two duplexes of 8 and 14 base pairs have been studied by 31P NMR and complete assignment of all phosphorus signals of the bound DNA duplexes are presented. While for the free DNA, all 31P signals display limited spectral dispersion (< 0.8 p.p.m.) for the bound duplexes, 31P resonances are spread over 2 p.p.m. Based on the previously published 3D structure of hSRY-HMG, with the 8 mer it is demonstrated that the upfield shifted resonances correspond to the site of partial intercalation of an isoleucine side chain into the DNA. Moreover, the observation of significant difference in linewidths between the two duplexes allows to estimate lifetime of the complexes from 31P-31P 2D exchange experiments.

Determination of three-bond 1H3'-31P couplings in nucleic acids and protein-nucleic acid complexes by quantitative J correlation spectroscopy.

A new sensitive two-dimensional quantitative J correlation experiment is described for measuring 3JH3'-P couplings in nucleic acids and protein-nucleic acid complexes. The method is based on measuring the change in intensity of the 1H-1H cross peaks in a constant-time 1H-1H COSY experiment which occurs in the presence and absence of 3JH3'-P dephasing during the constant-time evolution period. For protein-nucleic acid complexes where the protein is 13C-labeled but the nucleic acid is not, 12C-filtering is readily achieved by the application of a series of 13C purge pulses during the constant time evolution period without any loss of signal-to-noise of the nucleic acid cross peaks. The method is demonstrated for the Dickerson DNA dodecamer and a 19 kDa complex of the transcription factor SRY with a 14mer DNA duplex. The same approach should be equally applicable to numerous other problems, including the measurement of JH-Cd couplings in cadmium-ligated proteins, or 3JCH couplings in other selectively enriched compounds.

Managing an increasingly complex system.

A study of more than 170,000 health care workers (including 47,692 registered nurses [RNs]) in 138 acute care health care organizations revealed that the role of the RN is characterized by excessive numbers of activities, a loss of focus on the professional components of nursing and significant activity overlap with other job classes. Additionally, the study found that these characteristics were related to reduced morale, decreased patient and physician satisfaction with care and increased health care costs. The results of this study suggest a need for nursing leaders to develop new methods for controlling the complexity of health care systems, particularly the complexity of the RN role. Controlling complexity requires better tools for identifying system inefficiencies, more advanced skills in cross-functional work process diagnostics and more effective strategies for reducing complexity across health care systems.

Identification of a novel stage of ribosome/nascent chain association with the endoplasmic reticulum membrane.

Protein translocation in the mammalian endoplasmic reticulum (ER) occurs cotranslationally and requires the binding of translationally active ribosomes to components of the ER membrane. Three candidate ribosome receptors, p180, p34, and Sec61p, have been identified in binding studies with inactive ribosomes, suggesting that ribosome binding is mediated through a receptor-ligand interaction. To determine if the binding of nascent chain-bearing ribosomes is regulated in a manner similar to inactive ribosomes, we have investigated the ribosome/nascent chain binding event that accompanies targeting. In agreement with previous reports, indicating that Sec61p displays the majority of the ER ribosome binding activity, we observed that Sec61p is shielded from proteolytic digestion by native, bound ribosomes. The binding of active, nascent chain bearing ribosomes to the ER membrane is, however, insensitive to the ribosome occupancy state of Sec61p. To determine if additional, Sec61p independent, stages of the ribosome binding reaction could be identified, ribosome/nascent chain binding was assayed as a function of RM concentration. At limiting RM concentrations, a protease resistant ribosome-membrane junction was formed, yet the nascent chain was salt extractable and cross-linked to Sec61p with low efficiency. At nonlimiting RM concentrations, bound nascent chains were protease and salt resistant and cross-linked to Sec61p with higher efficiency. On the basis of these and other data, we propose that ribosome binding to the ER membrane is a multi-stage process comprised of an initial, Sec61p independent binding event, which precedes association of the ribosome/nascent chain complex with Sec61p.

Localization of a novel tumor suppressor locus on human chromosome 3q important in osteosarcoma tumorigenesis.

Mitotic recombination org nondysjunction are common mechanism for tumor-specific loss of constitutional heterozyosity (LOH) and tumor suppressor allelic inactivation and can be useful in localizing new putative tumor suppressor genes. In osteosarcoma, the highest frequencies of LOH have been reported for chromosomes 3q, 13q, 17p, and 18q. The high incidence of LOH on chromosome 3q suggests the presence of a novel tumor suppressor gene. To localize this putative tumor suppressor gene, we have used polymorphic markers on chromosome 3q to define the minimal region in which mitotic recombination or deletion results in LOH, which should contain the tumor suppressor gene. This putative tumor suppressor has been localized to a region between 3q26.2-3q26.3 of less that 1 cM between the polymorphic loci D3S1212 and D3S1246.

Role of Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE) in the propagation of MPMV vectors by genetic complementation using homologous/heterologous env genes.

To study Mason-Pfizer monkey virus (MPMV) replication over a single round, virus particles were generated that contain a replication-defective vector encoding a dominant selectable marker, the hygromycin B phosphotransferase (hyg) gene. Genetic complementation with a homologous MPMV envelope glycoprotein (Env-gp) or pseudotyping by several heterologous Env-gps from a variety of viruses resulted in infectious MPMV particles containing the replication-defective RNA. Recently, it has been shown that human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) Rev and Rev-responsive element (RRE) functions can be substituted in vitro by a cis-acting sequence, the constitutive transport element (CTE), from simian type D retroviruses like MPMV and simian retrovirus type 1 (SRV-1). To determine whether CTE of MPMV is necessary for MPMV nucleic acid propagation, an MPMV vector that lacked the terminally located CTE was generated. Propagation of this vector was completely abrogated in the absence of CTE, showing the importance of CTE in MPMV replication. Insertion of CTE back into the MPMV genome in the sense orientation rescued replication to wild-type levels. Slot-blot analysis of nuclear versus cytoplasmic RNA fractions revealed that most of the messages were sequestered in the nucleus of cells transfected with the CTE(-) vectors and very little was transported to the cytoplasm. To test whether HIV-1 or SIV RREs could complement CTE function, the HIV-1 or SIV RREs were inserted in the CTE(-) vectors, trans complementation of CTE(-)RRE(+) vectors with Env-and Rev-expression plasmids rescued propagation of the CTE(-) vectors. Computer analysis predicted an RNA secondary structure in MPMV CTE analogous to the HIV-1 and SIV RREs that could form three stable stem loops, the first of which contains a site similar to the Rev-binding domain in the HIV-1 RRE. The presence of a higher-order CTE structure was analyzed by mutational analysis. We conclude that CTE is important in the replication of MPMV and affects the nucleocytoplasmic transport and/or stability of viral messages similar to the Rev/RRE regulatory system of HIV-1 and SIV.

Thermodynamic effects of mutations on the denaturation of T4 lysozyme.

We investigated the folding of substantially destabilized mutant forms of T4 lysozyme using differential scanning calorimetry and circular dichroism measurements. Three mutations in an alpha-helix in the protein's N-terminal region, the alanine insertion mutations S44[A] and K48[A], and the substitution A42K had previously been observed to result in unexpectedly low apparent enthalpy changes of melting, compared to a pseudo-wild-type reference protein. The pseudo-wild-type reference protein thermally unfolds in an essentially two-state manner. However, we found that the unfolding of the three mutant proteins has reduced cooperativity, which partially explains their lower apparent enthalpy changes. A three-state unfolding model including a discrete intermediate is necessary to describe the melting of the mutant proteins. The reduction in cooperativity must be considered for accurate calculation of the energy changes of folding. Unfolding in two stages reflects the underlying two-subdomain structure of the lysozyme protein family.

Clinical reengineering. Integrating medical and operational management.

In the continuing push for cost containment in health care, many organizations have turned to cost reduction methods that fundamentally change the way care is delivered. As health care organizations continue to make financially-driven staffing changes that impact patient care, medical leadership must take on greater responsibility for operational management. Physician executives are uniquely qualified to take on leadership roles in work redesign, and must do so to ensure excellent and fiscally-responsible patient care. This article presents a proven methodology for work redesign that helps physician executives apply their clinical skills to operational management in designing new health care delivery models.

The Moloney murine sarcoma virus ts110 5' splice site signal contributes to the regulation of splicing efficiency and thermosensitivity.

The 5' splice site signal (5'ss) in Moloney murine sarcoma virus ts110 (MuSVts110) RNA was found to participate in the regulation of its splicing phenotype. This 5'ss (CAG/GUAGGA) departs from the mammalian consensus (CAG/GURAGU) at positions +4 and +6, both of which base pair with U1 and U6 small nuclear RNAs during splicing. A doubling in splicing efficiency and near elimination of the splicing thermosensitivity characteristic of MuSVts110 were observed in 5'ss mutants containing a U at position +6 (termed 5' A6U), even in those in which U1-5'ss complementarity had been reduced. At the permissive temperature (28 degrees C), the 5' A6U mutation increased the efficiency of the second splicing reaction, while at the nonpermissive temperature (39 degrees C), both splicing reactions were positively affected.

Cutting healthcare costs through work force reductions. Studies find what works and what doesn't.

Restructuring to cut costs often involves work force reductions. Two recent studies of restructuring in healthcare organizations have found that how an organization reduces its work force is just as important as whether it does. Organizations that implemented across-the-board staff cuts achieved limited cost savings. They also experienced decreased clinical quality, reduced patient satisfaction, and increased staff turnover. However, organizations that reduced their work forces only after a thorough assessment of work processes and job roles achieved greater cost savings, improved clinical quality, higher patient satisfaction, and less staff turnover.

Branchpoint and polypyrimidine tract mutations mediating the loss and partial recovery of the Moloney murine sarcoma virus MuSVts110 thermosensitive splicing phenotype.

Balanced splicing of retroviral RNAs is mediated by weak signals at the 3' splice site (ss) acting in concert with other cis elements. Moloney murine sarcoma virus MuSVts110 shows a similar balance between unspliced and spliced RNAs, differing only in that the splicing of its RNA is, in addition, growth temperature sensitive. We have generated N-nitroso-N-methylurea (NMU)-treated MuSVts110 revertants in which splicing was virtually complete at all temperatures and have investigated the molecular basis of this reversion on the assumption that the findings would reveal cis-acting elements controlling MuSVts110 splicing thermosensitivity. In a representative revertant (NMU-20), we found that complete splicing was conferred by a G-to-A substitution generating a consensus branchpoint (BP) signal (-CCCUGGC- to -CCCUGAC- [termed G(-25)A]) at -25 relative to the 3' ss. Weakening this BP to -CCCGAC- [G(-25)A,U(-27)C] moderately reduced splicing at the permissive temperature and sharply inhibited splicing at the originally nonpermissive temperature, arguing that MuSVts110 splicing thermosensitivity depends on a suboptimal BP-U2 small nuclear RNA interaction. This conclusion was supported by results indicating that lengthening the short MuSVts110 polypyrimidine tract and altering its uridine content doubled splicing efficiency at permissive temperatures and nearly abrogated splicing thermosensitivity. In vitro splicing experiments showed that MuSVts110 G(-25)A RNA intermediates were far more efficiently ligated than RNAs carrying the wild-type BP, the G(-25)A,U (-27)C BP, or the extended polypyrimidine tract. The efficiency of ligation in vitro roughly paralleled splicing efficiency in vivo [G(-25)A BP > extended polypyrimidine tract > G(-25)A,U(-27)C BP > wild-type BP]. These results suggest that MuSVts110 RNA splicing is balanced by cis elements similar to those operating in other retroviruses and, in addition, that its splicing thermosensitivity is a response to the presence of multiple suboptimal splicing signals.

Stage- and ribosome-specific alterations in nascent chain-Sec61p interactions accompany translocation across the ER membrane.

Near-neighbor interactions between translocating nascent chains and Sec61p were investigated by chemical cross-linking. At stages of translocation before signal sequence cleavage, nascent chains could be cross-linked to Sec61p at high (60-80%) efficiencies. Cross-linking occurred through the signal sequence and the mature portion of wild-type and signal cleavage mutant nascent chains. At later stages of translocation, as represented through truncated translocation intermediates, cross-linking to Sec61p was markedly reduced. Dissociation of the ribosome into its large and small subunits after assembly of the precursor into the translocon, but before cross-linking, resulted in a dramatic reduction in subsequent cross-linking yield, indicating that at early stages of translocation, nascent chain-Sec61p interactions are in part mediated through interactions of the ribosome with components of the ER membrane, such as Sec61p. Dissociation of the ribosome was, however, without effect on subsequent translocation. These results are discussed with respect to a model in which Sec61p performs a function essential for the initiation of protein translocation.

Rightsizing healthcare: what works, what doesn't, and why.

Recently a nationwide study of the methods and consequences of restructuring in healthcare was completed by E.C. Murphy, Ltd., in cooperation with the American Society for Work Redesign. The results have important implications for hospitals and systems involved in or considering restructuring. The following are excerpts of an interview with Emmett C. Murphy, PhD, president of the firm.

Probing the mechanism of staphylococcal nuclease with unnatural amino acids: kinetic and structural studies.

Staphylococcal nuclease is an enzyme with enormous catalytic power, accelerating phosphodiester bond hydrolysis by a factor of 10(16) over the spontaneous rate. The mechanistic basis for this rate acceleration was investigated by substitution of the active site residues Glu43, Arg35, and Arg87 with unnatural amino acid analogs. Two Glu43 mutants, one containing the nitro analog of glutamate and the other containing homoglutamate, retained high catalytic activity at pH 9.9, but were less active than the wild-type enzyme at lower pH values. The x-ray crystal structure of the homoglutamate mutant revealed that the carboxylate side chain of this residue occupies a position and orientation similar to that of Glu43 in the wild-type enzyme. The increase in steric bulk is accommodated by a backbone shift and altered torsion angles. The nitro and the homoglutamate mutants display similar pH versus rate profiles, which differ from that of the wild-type enzyme. Taken together, these studies suggest that Glu43 may not act as a general base, as previously thought, but may play a more complex structural role during catalysis.

How to design a horizontal patient-focused hospital.

Work Imaging is an executive information system for analyzing the cost effectiveness and efficiency of work processes and structures in health care. Advanced Work Imaging relational database technology allows managers and employees to take a sample work activities profile organization-wide. This is married to financial and organizational data to produce images of work within and across all functions, departments, and levels. The images are benchmarked against best practice data to provide insight on the quality and cost efficiency of work practice patterns, from individual roles to departmental skill mix to organization-wide service processes.

Regulation of the efficiency and thermodependence of murine sarcoma virus MuSVts110 RNA splicing by sequences in both exons.

Efficient splicing of MuSVts110 RNA is restricted to temperatures of 33 degrees or lower. Previously, we have shown that this conditional splicing event is mediated, in part, by cis-acting intronic sequences. We have now examined the role of exon sequences in MuSVts110 RNA splicing. We found that deletion of all but 36 nucleotides of the gag exon (E1) yielded a transcript incapable of supporting splicing. However, inefficient, growth temperature-dependent splicing was recovered after restoration of the 300 nucleotides of E1 proximal to the 5' splice site (5' ss). Increasingly efficient splicing was observed as more E1 was restored. Hence, although MuSVts110 E1 sequences were required for splicing, they were not involved in its thermodependence. Similarly, removal of all but 88 nucleotides of the mos exon (E2) abolished splicing at the usual 3' splice site (3' ss). In contrast to E1, restoration of the 200 nucleotides of E2 adjacent to the 3' ss reactivated efficient, temperature-independent splicing. Thermodependent splicing, however, reappeared with the replacement of E2 sequences located more than 400 nucleotides distal to the 3' splice site. In MuSVts110 mutants containing the minimum amounts of both E1 and E2 which would support splicing, splicing was both far more efficient than predicted and temperature-independent, suggesting that cooperation between E1 and E2 may help to regulate MuSVts110 splicing.

Extraparenchymal neurocysticercosis: report of five cases and review of management.

Neurocysticercosis due to parenchymal cysts carries a good prognosis regardless of therapy. Extraparenchymal neurocysticercosis (including ventricular, spinal, and subarachnoid types) carries a poorer prognosis. Most extraparenchymal cases present with hydrocephalus. Medical treatment alone in doses and schedules developed for parenchymal disease is frequently unsuccessful. For ventricular disease, most cases can be managed with shunting procedures either alone or together with the administration of antiparasitic agents (e.g., praziquantel or albendazole), without extirpation of the cysts. Subarachnoid disease was formerly associated with a case fatality rate of about 50%. However, with the combination of shunting procedures for hydrocephalus, antiparasitic agents, and, in some cases, surgical extirpation of the cysts, the prognosis is much improved. Spinal cysticercosis can be either leptomeningeal (which responds like subarachnoid disease) or intramedullary. For all forms of neurocysticercosis, the role of antiparasitic agents needs to be better defined.