Snapshots of catalysis: the structure of fructose-1,6-(bis)phosphate aldolase covalently bound to the substrate dihydroxyacetone phosphate.

Fructose-1,6-bis(phosphate) aldolase is an essential glycolytic enzyme found in all vertebrates and higher plants that catalyzes the cleavage of fructose 1,6-bis(phosphate) (Fru-1,6-P(2)) to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). Mutations in the aldolase genes in humans cause hemolytic anemia and hereditary fructose intolerance. The structure of the aldolase-DHAP Schiff base has been determined by X-ray crystallography to 2.6 A resolution (R(cryst) = 0.213, R(free) = 0.249) by trapping the catalytic intermediate with NaBH(4) in the presence of Fru-1,6-P(2). This is the first structure of a trapped covalent intermediate for this essential glycolytic enzyme. The structure allows the elucidation of a comprehensive catalytic mechanism and identification of a conserved chemical motif in Schiff-base aldolases. The position of the bound DHAP relative to Asp33 is consistent with a role for Asp33 in deprotonation of the C4-hydroxyl leading to C-C bond cleavage. The methyl side chain of Ala31 is positioned directly opposite the C3-hydroxyl, sterically favoring the S-configuration of the substrate at this carbon. The "trigger" residue Arg303, which binds the substrate C6-phosphate group, is a ligand to the phosphate group of DHAP. The observed movement of the ligand between substrate and product phosphates may provide a structural link between the substrate cleavage and the conformational change in the C-terminus associated with product release. The position of Glu187 in relation to the DHAP Schiff base is consistent with a role for the residue in protonation of the hydroxyl group of the carbinolamine in the dehydration step, catalyzing Schiff-base formation. The overlay of the aldolase-DHAP structure with that of the covalent enzyme-dihydroxyacetone structure of the mechanistically similar transaldolase and KDPG aldolase allows the identification of a conserved Lys-Glu dyad involved in Schiff-base formation and breakdown. The overlay highlights the fact that Lys146 in aldolase is replaced in transaldolase with Asn35. The substitution in transaldolase stabilizes the enamine intermediate required for the attack of the second aldose substrate, changing the chemistry from aldolase to transaldolase.

Method for phosphorothioate antisense DNA sequencing by capillary electrophoresis with UV detection.

The progress of antisense DNA therapy demands development of reliable and convenient methods for sequencing short single-stranded oligonucleotides. A method of phosphorothioate antisense DNA sequencing analysis using UV detection coupled to capillary electrophoresis (CE) has been developed based on a modified chain termination sequencing method. The proposed method reduces the sequencing cost since it uses affordable CE-UV instrumentation and requires no labeling with minimal sample processing before analysis. Cycle sequencing with ThermoSequenase generates quantities of sequencing products that are readily detectable by UV. Discrimination of undesired components from sequencing products in the reaction mixture, previously accomplished by fluorescent or radioactive labeling, is now achieved by bringing concentrations of undesired components below the UV detection range which yields a 'clean', well defined sequence. UV detection coupled with CE offers additional conveniences for sequencing since it can be accomplished with commercially available CE-UV equipment and is readily amenable to automation.

Factors associated with changes in occupancy rates of California short-term hospitals: an analysis of statewide hospital discharge data.

Decline in per cent occupancy of California short-term hospitals between 1969 and 1972 was due principally to an increase in bed supply that exceeded population growth. A lesser contributory cause--decline in utilization--was due entirely to decreases in length-of-stay. Analysis of data from Statewide one-week discharge surveys, carried out in 1968 and 1970, indicates the decrease in average stay was largely centered in Medicare and Medi-Cal (Medicaid) pay classes. Admissions, however, did not decrease. These declines in stay predated the introduction of special increased restrictions on access to medical care into the Medi-Cal program in April 1970. Principal results of the study indicate that in California, government supervision of payment for hospital care has operated to reduce length-of-stay, but thus far does not seem to have affected admission rates. Insofar as these results may hold for other States, they seem to implay certain conditions that may be expected to occur with an extension of National Health insurance to the general population. Chief among these is a further depression of occupancy ratios if bed supply is held constant or increases relative to population. They also imply that further substantial reduction in hospital utilization under Medicare and Medicaid must be sought in admission rates rather than length-of-stay.

Cost-sharing and prior authorization effects on Medicaid services in California: Part II: The providers' reactions.

Providers for California's Medical program were interviewed to determine their reaction to the copayment experiment. Private physicians, pharmacists, and nursing homes routinely collected the copayments and though it was not a particular hardship on the patients. Hospital outpatient departments often did not collect. Physicians both in hospitals and in private practice generally did not know which patients were copayers and hence copayment had no effect on their practice. Pharmacists reported some delay and some selectivity on the part of patients in the filling of their prescriptions. All providers felt as much more harassment and interference from the concurrent prior authorization requirements. This effect may have swamped copayment effects.

Cost-sharing and prior authorization effects on Medicaid services in California: part I. The beneficiaries' reactions.

In January 1972, California initiated a cost-sharing "experiment" as a means of containing rising costs of the Medicaid program. Beneficiaries who had some personal assets were required to pay a token amount for each doctor visit or prescription. This was expected to deter unnecessary services without affecting really needed care. An interview survey was made of beneficiaries continuously in copayment status throughout the 18 months of the experiment. Most beneficiaries had poor knowledge of the program, and tended to confuse copayment with the concurrent constraint of prior authorization for certain classes of service. The beneficiaries reported that the copayment was almost always collected, and most thought it had not affected their health care, but a significant 17 per cent throught it had reduced the care available to them, and these 17 per cent were for the most part in households with high medical need.

Copayments for ambulatory care: penny-wise and pound-foolish.

The California "copayment experiment" imposed a charge of $1 on certain Medicaid beneficiaries for the first two visits to a doctor and 50 cents for the first two drug prescriptions each month, effective January 1, 1972. Data on utilization rates were gathered for six months before this date and for 12 months after it. While other administrative requirements, like prior authorization of certain services, doubtless also played a part, it was found that, following the start of copayment, utilization of ambulatory doctor's office visits and other services associated with them showed a decline, relative to that of the non-copayment cohort. After a brief lag, however, hospitalization rates in the copay cohort rose to levels higher than those of the non-copayment cohort-more than offsetting the savings to the state from the reduction of ambulatory service use rates. Due presumably to the neglect of early medical care because of the inhibiting effect of the copayments, these higher use rates of costly hospitalizations suggest that financial deterrents on access to ambulatory service by poor people are penny-wise and pound-foolish, not to mention their effects on health and well-being.

Quality of medical care: a factor analysis approach using medical records.

A relatively inexpensive, reliable, and unobtrusive method is described for measuring the content of medical care. Factor analysis of the content of the records of more than 11,000 physician-patient encounters from six different health insurance plans extracted four main factors or dimensions that together explained 42 percent of the variance in record content. Appropriate names for these dimensions appear to be: "prevention," "rationality," "verification," and "continuity." The method is tested by scoring the six insurance plans on the four factors.