4-oxo-4H-quinolizine-3-carboxylic acids as Mg2+ selective, fluorescent indicators.

In this paper, we present the synthesis of a new series of substituted 4-oxo-4H-quinolizine-3-carboxylic acids and the evaluation of their fluorescent response upon complexation with Mg2+ and Ca2+. This has led to the development of the first Mg2+-selective, ratioable fluorescent indicators. We found that 4-oxo-4H-quinolizine-3-carboxylates are excellent fluorophores which show a strong fluorescent response to Mg2+ but not to Ca2; the latter metal ion often causing interference during Mg2+ measurements with previously described indicators. The dissociation constants of the studied fluorophores-around 1.0 mM-are of the same order of magnitude as is usually observed for intracellular Mg2+.

Interligand Overhauser effects in type II dihydrofolate reductase.

R67 dihydrofolate reductase (DHFR) is a type II DHFR produced by bacteria as a resistance mechanism to the increased clinical use of the antibacterial drug trimethoprim. Type II DHFRs are not homologous in either sequence or structure with chromosomal DHFRs. The type II enzymes contain four identical subunits which form a homotetramer containing a single active site pore accessible from either end. Although the crystal structure of the complex of R67 DHFR with folate has been reported [Narayana et al. (1995) Nat. Struct. Biol. 2, 1018], the nature of the ternary complex which must form with substrate and cofactor is unclear. We have performed transferred NOE and interligand NOE (ILOE) studies to analyze the ternary complexes formed from NADP(+) and folate in order to probe the structure of the ternary complex. Consistent with previous studies of the binary complex formed from another type II DHFR, the ribonicotinamide bond of NADP(+) was found to adopt a syn conformation, while the adenosine moiety adopts an anti conformation. Large ILOE peaks connecting NADP(+) H4 and H5 with folate H9 protons are observed, while the absence of a large ILOE connecting NADP(+) H4 and H5 with folate H7 indicates that the relative orientation of the two ligands differs significantly from the orientation in the chromosomal enzyme. To obtain more detailed insight, we prepared and studied the folate analogue 2-deamino-2-methyl-5,8-dideazafolate (DMDDF) which contains additional protons in order to provide additional NOEs. For this analogue, the exchange characteristics of the corresponding ternary complex were considerably poorer, and it was necessary to utilize higher enzyme concentrations and higher temperature in order to obtain ILOE information. The results support a structure in which the NADP(+) and folate/DMDDF molecules extend in opposite directions parallel to the long axis of the pore, with the nicotinamide and pterin ring systems approximately stacked at the center. Such a structure leads to a ternary complex which is in many respects similar to the gas-phase theoretical calculations of the dihydrofolate-NADPH transition state by Andres et al. [(1996) Bioorg. Chem. 24, 10-18]. Analogous NMR studies performed on folate, DMDDF, and R67 DHFR indicate formation of a ternary complex in which two symmetry-related binding sites are occupied by folate and DMDDF.

A new approach to the synthesis of APTRA indicators.

We have devised a general synthesis of Mg(2+) indicators which is based on the aminophenol triacetic acid (APTRA) structure. The key step is a palladium-catalyzed coupling reaction of a precursor of the APTRA ligand with a fluorescent group. This strategy resulted in new ratioable fluorescent APTRA indicators and the finding that the fluorescence response of these indicators is different for Mg(2+) and Ca(2+) in some cases. We believe that this represents a generally useful approach for combining fluorophore and chelator functionalities.

Epidemiology of onychomycosis in special-risk populations.

A person's susceptibility to onychomycosis and the course of the disease once the nails are infected are functions of the interaction of the fungal agent, the host, and environmental factors. The disease is reported to have an overall prevalence of 2% to 13%, but the prevalence is much higher in certain populations, such as older people and those with immunosuppressive conditions. Although onychomycosis may be merely a nuisance and an embarrassment for healthy individuals, some morbidity is seen with all population groups, but especially high-risk patients: diabetics, patients infected with human immunodeficiency virus (HIV), patients with acquired immunodeficiency syndrome (AIDS), and patients with other types of immunosuppression (eg, transplant recipients and patients on long-term corticosteroid therapy). Whether the increased prevalence of onychomycosis in the elderly (up to 30% by age 60) is related to changes in immune function is not known.

Podiatric medical education and practice. 1960s to the 21st century.

An overview of the development of medical and podiatric medical education in the US is provided. Beginning with the 1910 Flexner Report in medicine and moving to the 1996 Educational Enhancement Project of podiatric medicine, major changes have taken place that have made podiatric medicine a profession quite comparable to medicine. This is most obvious in the education and training process. It is essential that both the content and method of educating the podiatric physician for the 21st century reflect the rapidly evolving health care delivery and financing system.

Partnership between a Veterans Affairs medical center and a podiatric medical college.

The health care academic delivery system is dramatically changing in today's economy. In order to survive, the delivery system must decrease its costs and increase productivity. Integration of academic affiliates and community health care facilities has produced a more efficient health care system and improved medical education. The formation and methodology of the mutual benefits and responsibilities between a health care system and a college of podiatric medicine are examined in detail. Developing unique sharing partnerships can mutually improve medical student experiences, reduce financial burdens, combine joint research projects, and ultimately improve patient care.

Measurement of free Ca2+ in sarcoplasmic reticulum in perfused rabbit heart loaded with 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid by 19F NMR.

Measurements of free calcium ion concentration in the sarcoplasmic reticulum ([Ca2+]SR) and an evaluation of its relationship to changes in cytosolic free calcium and energy state of the cell, as well as heterogeneity of the SR calcium pool, were performed using 19F NMR in Langendorff perfused rabbit hearts loaded with acetoxymethyl ester of 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid. We report a base-line time-average [Ca2+]SR value of 1.5 mM (n = 13) in the beating heart, similar to the value measured at diastole. We further report that [Ca2+]SR decreases by approximately 30% at the start of systole and that there is no evidence of spacial heterogeneity in [Ca2+]SR during the contraction cycle. However, there appears to be a heterogeneous response to SR calcium channel release activator (caffeine) and SR calcium-ATPase inhibitor (cyclopiazonic acid), consistent with studies suggesting that there are subpopulations of SR. Raising cytosolic free calcium by depolarizing the cell with 30 mM extracellular KCl, resulted in an increase in [Ca2+]SR; however, the calcium gradient was unchanged. Lowering cell phosphorylation potential, which would reduce the free energy available for the SR Ca2+-ATPase, leads to a decrease in the calcium gradient across the SR, but this reduced gradient was primarily due to an increase in cytosolic free calcium and not a net release of SR calcium.

History and epidemiology of acquired immune deficiency syndrome.

Acquired immune deficiency syndrome (AIDS) and human immunodeficiency virus (HIV) were first noticed in the US in 1981 and continue to spread today. Initially a disease associated completely with homosexual males, it is increasing in incidence and prevalence among heterosexual males and females, particularly, but not limited to, injection drug users. This disease is much more prevalent among blacks and Hispanics. Podiatric physicians are at risk of acquiring the disease as a result of their frequent use of injections and surgical intervention, particularly involving bone. In addition, the foot is a potential portal of entry for HIV infection because of contamination by blood on the feet of podiatric surgeons and their assistants during surgery.

Fluorinated o-aminophenol derivatives for measurement of intracellular pH.

The simple 2-aminophenol group which serves as a building block for many cationic indicators has been modified to yield a series of pH sensitive probes. This approach is based on the replacement of one of the N-acetate groups of the chelator APTRA (o-aminophenol N,N,O-triacetate) by an N-ethyl group. The resulting series of (N-ethylamino)phenol (NEAP) compounds exhibit pK values in the physiological range and negligible affinity for physiological levels of other ions. Three fluorinated analogs have been prepared: N-ethyl-5-fluoro-2-aminophenol N,O-diacetate (5F NEAP), N-ethyl-2-((2-fluoro-4-carboxybenzyl)oxy)-4-fluoroaniline-N- acetic acid (5F NEAP-2), and 1-(2-(N-ethylamino)-5-fluorophenoxy)-2-(2-fluoro-4- aminophenoxy)ethane-N,N',N'-triacetic acid (5F NEAP-3). These derivatives exhibit total titration shifts of approximately 11 ppm. NEAP-2 and NEAP-3 contain an additional fluorine to serve as an internal chemical shift reference, and NEAP-3, the most highly charged analog prepared, was designed in order to minimize leakage.

NMR-sensitive fluorinated and fluorescent intracellular calcium ion indicators with high dissociation constants.

A new series of high-dissociation constant (KD) Ca2+ indicators has been developed to reduce perturbations due to buffering of transients, to carry out measurements in cells and organelles with high basal Ca2+ concentrations, and to measure cytosolic Ca2+ levels in the presence of perturbations that may significantly increase these levels. A tetrafluorinated derivative of the chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (TF-BAPTA), has a KD of 65 microM and exhibits two fluorine nuclear magnetic resonances, one of which is insensitive to Ca2+ chelation and the second of which shifts by approximately 10 ppm upon Ca2+ binding. TF-BAPTA has pK values of approximately 5.0 and Mg2+ dissociation constants > 50 mM. At a field of 8.5 T, the Ca(2+)-sensitive resonance is in fast-intermediate exchange. Correction factors for the effects of intermediate exchange and for the effect of protonation (pK approximately 5.0) and Mg2+ complexation are discussed. An analogous approach has been used to synthesize 2-[2-(5-carboxyoxazole)]-5-[2-(2-bis(carboxymethyl) amino-5,6-difluorophenoxy)]ethoxy-6-bis(carboxymethyl)aminobenz ofuran (fura F), a structural analogue of fura 2, which exhibits fluorescence characteristics similar to those of fura 2, but has a KD of 20 microM.

Measurement of cytosolic free calcium in perfused rat heart using TF-BAPTA.

The feasibility and usefulness of loading 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (TF-BAPTA), a new high-dissociation constant (KD) (65 microM) Ca2+ indicator, into perfused rat heart is demonstrated. TF-BAPTA-loaded perfused rat heart showed less than a 10% reduction in left ventricular developed pressure. In addition, loading perfused rat heart with TF-BAPTA had no effect on cell high-energy phosphates measured by 31P-nuclear magnetic resonance (NMR). Cytosolic free Ca2+ (Ca2+i) can be monitored in TF-BAPTA-loaded perfused rat heart using 19F-NMR. TF-BAPTA has a Ca(2+)-insensitive resonance (6F) and a Ca(2+)-sensitive fluorine (5F) that responds to changes in Ca2+ binding with fast exchange kinetics at magnetic fields < or = 8.5 T. Thus the shift difference between the 5F and 6F resonances is a measure of Ca2+i. Given the high KD and the slight differences in intra- vs. extracellular fluorine shifts, TF-BAPTA is not well suited for measuring basal Ca2+i, but it is useful for measuring increases in Ca2+i above this level. For studies in which intracellular pH changes are significant, e.g., during ischemia, pH-dependent corrections must be made to obtain an accurate Ca2+i value. Given its fast exchange kinetics, TF-BAPTA is also useful for measurement of free Ca2+ in different compartments or cells with different Ca2+i. We show that the rise in Ca2+i is not uniform during prolonged global ischemia (60 min); several different Ca2+i values are present. Thus TF-BAPTA is a useful new indicator for measuring elevations in Ca2+i or compartmentation of Ca2+i.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary care podiatric medicine and entry level residency. The new millennium.

The author presents the perspective that the nation's health care initiatives demand that greater attention be given to primary care providers. Inasmuch as the credibility of the podiatric medical profession must function in a health care environment dominated by allopathic and osteopathic physicians, the podiatric primary care initiative must be pursued within the guidelines and definitions for primary care that are present in all of mainstream medicine. The author argues that primary care podiatric medicine must establish itself as a specialty that stands as an equal along side of the other recognized specialties in podiatric medicine. Also, in keeping with the essential educational needs for specialty training, the development of residencies in primary podiatric medicine is crucial to assuring a credible area of special practice.

Intracellular pH modulates cytosolic free magnesium in cultured chicken heart cells.

To assess the role of pH in cellular Mg homeostasis, cytosolic pH (pHi) was manipulated by the NH4Cl prepulse technique; pHi, cytosolic Mg2+ (Mgi), and cytosolic Ca2+ (Cai) were measured fluorometrically in single cultured embryonic chicken heart cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), FURAPTRA, and fura-2, respectively. The basal values obtained were as follows: pHi = 7.21 +/- 0.10 (n = 7), [Mg]i = 0.51 +/- 0.08 mM (n = 9), [Ca]i = 126 +/- 15 nM (n = 7). When cells were perfused with 10 mM NH4Cl solution for 5 min, a transient alkalinization (0.53 U) of the cytosol was accompanied by a transient decrease (0.12 mM) in [Mg]i and a transient increase (59 nM) in [Ca]i; these changes approached control levels within 5 min. Upon removal of NH4Cl, a transient acidification (0.89 U) of the cytosol was accompanied by a transient increase (0.10 mM) in [Mg]i and a transient increase (125 nM) in [Ca]i; again, these changes returned toward control levels within 5 min. No significant changes in total cell Mg or Ca were observed during these manipulations. NH4Cl-evoked changes in [Mg]i were not altered significantly by either Mg-free or Ca-free conditions. Changes in [Mg]i were inversely correlated with changes in pHi and were not secondary to changes in [Ca]i. The results suggest that pHi modulates Mgi, probably by affecting cytosolic Mg binding and/or the transport of Mg across subcellular organelles.

Smoking and peripheral vascular disease. Podiatric medical update.

Although there is strong evidence that cigarette smoking is perhaps the major risk factor associated with peripheral occlusive vascular disease, there still appears to be little indication that clinicians in podiatric medicine and patients recognize this. When smoking is combined with other risk factors such as diabetes mellitus, the probability of developing peripheral arterial disease is greatly increased. In addition, smoking appears to accelerate the natural history of the process of atherogenesis and thrombosis. Diabetic neuropathy seems to have a greater prevalence when the patient has a history of an increased number of pack years smoked. By eliminating smoking, patients can often receive considerable relief from intermittent claudication and other sequelae of occlusive vascular disease, including the avoidance of amputation of the lower extremity. Patient care routinely should include efforts to prevent individuals from smoking as well as advice to smokers to stop. Podiatrists need to become a more prominent source of information about the hazards of smoking and should freely share this knowledge with their patients.

Concomitant Creutzfeldt-Jakob and Alzheimer diseases.

We report a 69-year-old female with cerebral and cerebellar symptomatology of 15-month duration. At autopsy, both panencephalopathic Creutzfeldt-Jakob and plaque-predominant Alzheimer diseases were found. Plaque amyloid was exclusively of the beta/A4 type, but abundant abnormal protease-resistant protein was identified by Western blot analysis of brain extracts.