Efficient expression of the gene for spinach phosphoribulokinase in Pichia pastoris and utilization of the recombinant enzyme to explore the role of regulatory cysteinyl residues by site-directed mutagenesis

Phosphoribulokinase (PRK), unique to photosynthetic organisms, is regulated in higher plants by thioredoxin-mediated thiol-disulfide exchange in a light-dependent manner. Prior attempts to overexpress the higher plant PRK gene in Escherichia coli for structure-function studies have been hampered by sensitivity of the recombinant protein to proteolysis as well as toxic effects of the protein on the host. To overcome these impediments, we have spliced the spinach PRK coding sequence immediately downstream from the AOX1 (alcohol oxidase) promoter of Pichia pastoris, displacing the chromosomal AOX1 gene. The PRK gene is now expressed, in response to methanol, at 4-6% of total soluble protein, without significant in vivo degradation of the recombinant enzyme. This recombinant spinach PRK is purified to homogeneity by successive anion-exchange and dye-affinity chromatography and is shown to be electrophoretically and kinetically indistinguishable from the authentic spinach counterpart. Site-specific replacement of all of PRK's cysteinyl residues (both individually and in combination) demonstrates a modest catalytically facilitative role for Cys-55 (one of the regulatory residues) and the lack of any catalytic role for Cys-16 (the other regulatory residue), Cys-244, or Cys-250. Mutants with seryl substitutions at position 55 display non-hyperbolic kinetics relative to the concentration of ribulose 5-phosphate. Sulfate restores hyperbolic kinetics and enhances kinase activity, presumably reflecting conformational differences between the position 55 mutants and wild-type enzyme. Catalytic competence of the C16S-C55S double mutant proves that mere loss of free sulfhydryl groups by oxidative regulation cannot account entirely for the accompanying total inactivation.     

Factors influencing survival after gamma knife radiosurgery for patients with single and multiple brain metastases

BACKGROUND: Significant changes are restructurng the U.S. health care delivery system. National health reform is now extending itself into the public sector. Increased health and medical costs by federal and state governments are forcing a reevaluation of major entitlement programs, especially Medicaid. METHODS/RESULTS: Because Medicaid is the single largest item in many state budgets, states are now enrolling Medicaid patients into managed and coordinated care arrangements as a means to control costs and increase access to care. HMOs are not only competing for private patients but also actively seeking the Medicaid population. Nationally, almost one-fourth of all Medicaid patients are now enrolled in managed care plans. Various models and approaches have been developed by individual states. CONCLUSIONS: Because managed care enrollment in the Medicaid program has increased substantially in recent years, selected services including vision care are no longer rendered by any practitioner willing to accept Medicaid fees. Freedom of choice is now restricted to pre-selected and panel practitioners participating with the managed care program. The rules, regulations, billing procedures, fees, and program requisites will differ under managed care programs. Private optometric practitioners must consider entering economic and organizational relationships and linkages that make them attractive to managed care organizations.     

Ischio-pubic-patellar hypoplasia: is it a new syndrome?

Aplasia/hypoplasia of the patella has been described as an isolated finding or, more commonly, as a part of congenital syndromes. We describe here bilateral absence of the patella in an 11-year-old girl with absence of the ischial and inferior pubic rami bilaterally. Other associated skeletal and soft-tissue deformities are also reported. To our knowledge, the constellation of these findings has not been described previously and represents a unique syndrome.     

Arthritic temporomandibular joint: correlation of macromolecular contrast-enhanced MR imaging parameters and histopathologic findings

PURPOSE: To assess the utility of macromolecular contrast material-enhanced magnetic resonance (MR) imaging parameters for determining the histopathologic severity of temporomandibular joint (TMJ) arthritis. MATERIALS AND METHODS: Ovalbumin was used to induce arthritis in the TMJs of 10 previously sensitized adult white rabbits. Five rabbits composed the sham-treated control group. Dynamic spin-echo imaging was performed immediately before and for 30 minutes after injection of macromolecular contrast medium. Histologic specimens of TMJ were assessed quantitatively for arthritis. Changes in MR signal intensity were derived from the synovial and subsynovial tissues of the TMJ, and plasma volume (PV) and permeability surface area product (PS) were calculated. These MR parameters and the arthritic scores were compared between sham-treated and antigen-challenged TMJs. The relationships between MR parameters and histopathologic indexes were also determined. RESULTS: Arthritic TMJs showed marked enhancement of the synovial and subsynovial tissues over the imaging period. PS and all histopathologic indexes of arthritis were significantly greater (P < .005) in antigen-challenged than in sham-treated TMJs. PS demonstrated strong positive relationships with all histologic parameters of arthritis, indicating its utility for assessing the severity of joint inflammation. CONCLUSION: Macromolecular contrast-enhanced MR imaging enables quantification of PS and PV in inflamed joints. This technique may provide insights into the pathogenesis of joint inflammation and noninvasive monitoring of disease severity and treatment response in arthritis.     

Relationships of PPARgamma and PPARgamma2 mRNA levels to obesity, diabetes and hyperinsulinaemia in rhesus monkeys

OBJECTIVE: To examine the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) together with CCAAT/enhancer binding protein alpha (C/EBPalpha), lipoprotein lipase (LPL) and glucose transporter (GLUT4) mRNA in adipose tissue of rhesus monkeys in relation to obesity. DESIGN: Cloning of the PPARgamma1 and gamma2 cDNAs and analysis of PPARgamma, C/EBPalpha, LPL and GLUT4 mRNA levels in the adipose tissue of lean and obese monkeys. SUBJECTS: 28 rhesus monkeys (Macaca mulatta) with a wide range of body weights (9.2-22.6 kg) and with or without type 2 diabetes. MEASUREMENTS: Sequence of PPARgamma1 and gamma2. Tissue distribution of PPARgamma1 and gamma2. The mRNA levels of PPARgamma, C/EBPalpha, LPL and GLUT4 in adipose tissue. The ratio of PPARgamma2 mRNA to total PPARgamma mRNA. RESULTS: The monkey PPARgamma2 protein showed 99% identity with the human protein. PPARgamma1 mRNA was shown to be expressed in various tissues and most abundantly in adipose tissue. PPARgamma2 existed mainly in adipose tissue. A significant correlation between the ratio of PPARgamma2 mRNA to total PPARgamma mRNA and obesity was observed, whereas total PPARgamma mRNA levels showed no significant relationships to obesity. There was also a significant relationship between the ratio of PPARgamma2 mRNA to total PPARgamma mRNA and fasting plasma insulin concentration. The mRNA levels of C/EBPalpha, LPL and GLUT4 were highly correlated to that of total PPARgamma mRNA. They were also significantly correlated to the mRNA levels of PPARgamma1 and PPARgamma2. CONCLUSIONS: The ratio of PPARgamma2 mRNA to total PPARgamma mRNA is related to obesity in the rhesus monkey and mRNA expression of PPARgamma1, PPARgamma2, C/EBPalpha, LPL and GLUT4 appear to be coordinated in vivo.     

Restrictions on components of variance for epistatic models

The putative role(s) of a mechanically gated (MG) cation channel in Xenopus oocyte growth, maturation, fertilization and embryogenesis has been examined. Using a pharmacological approach, we have tested the effects of the MG channel blockers, gadolinium, gentamicin and amiloride on the above developmental events. Our results indicate that oocyte maturation, fertilization and early embryogenesis (up to the free-swimming stage 45) can proceed normally in the presence of concentrations of agents that either completely abolish (i.e., > or = 10 microM Gd3+) or partially block (i.e., 1 mM gentamicin) single MG channel activity as measured by patch-clamp recording. However, we also find that higher concentrations of Gd3+ (> or = 50 microM) can lead to an increased percentage (> 20%) of axis-perturbed embryos compared with control (< 1%) and that amiloride (0.5 mM) reduces the success of fertilization (from 100% to < 50%) and increases mortality (by approximately 75%) in developing embryos. Furthermore, we find that all three agents inhibit oocyte growth in vitro. However, their order of effectiveness (amiloride > gentamicin > Gd3+) is opposite to their order for blocking MG channels (Gd3+ > gentamicin > amiloride). These discrepancies indicated that the drugs effects occur by mechanisms other than, or in addition to, MG channel block. Our results provide no compelling evidence for the idea that MG channel activity is critical for development in Xenopus. This could mean that there are other mechanisms in the oocyte that can compensate when MG channel activity is blocked or that the protein that forms the channel can undergo additional interactions that result in a function insensitive to MG channel blockers.