Pharmaceutical services in the Department of Veterans Affairs.

The status of pharmaceutical services in the Department of Veterans Affairs (VA) is described. The VA health care system is transforming itself from a hospital-based organization into 22 health care networks that emphasize primary and ambulatory care. The impact on VA pharmacy has been substantial. Roles of VA pharmacists and technicians have been enhanced, and a clinical career ladder for pharmacists was created. VA pharmacy officials and leaders from the University of Tennessee College of Pharmacy have been partnering since 1988 in strategic planning to determine how VA pharmacy can do business and serve patients better. Areas targeted for implementation or improvement include staff development, prescribing authority for pharmacists, automation, the physical design of VA pharmacies, residency programs, and a pharmacy benefit management (PBM) product line. The VA PBM is working to enhance the appropriate use of pharmaceuticals in the veteran population, reduce overall health care expenditures, and provide a more consistent quality of care. Specific PBM programs involve the implementation of drug treatment guidelines, a national formulary, and national contracts. There are plans for pharmacoepidemiologic and pharmacoeconomic research in the geriatric veteran population. The VA health care system and its pharmacy service are changing in ways intended to bring about easier access to care, higher quality, and increased responsiveness to patients' needs.

Expression of rat neuronal nitric oxide synthase in Saccharomyces cerevisiae.

The rat neuronal nitric oxide synthase (nNOS) cDNA was expressed in Saccharomyces cerevisiae under the control of a hybrid PGK/GAL promoter, PAL. Galactose induction resulted in the production of a soluble 160 kDa protein that was recognised by anti-neuronal NOS antibody. NOS activity was detected by the conversion of [3H]arginine to [3H]citrulline and required the addition of the cofactors, calmodulin and tetrahydrobiopterin. The activity was inhibited by the arginine analogues NG-nitro-L-arginine and NG-nitro-L-arginine methyl ester.

Comparison of Department of Veterans Affairs pharmacy services in 1992 and 1994 with strategic-planning goals.

Data were collected from Department of Veterans Affairs (VA) medical center pharmacies in 1992 and 1994 to measure progress toward implementation of the VA 1990 strategic plan. A questionnaire was pretested and mailed to pharmacy chiefs at all 173 VA medical centers (VAMCs) with pharmacies in 1992. The same questionnaire, with slight modifications consistent with revision of the strategic plan, was mailed in 1994. Usable responses were received from more than 80% of VAMCs in both years. The number and types of activities, services, and staffing at VAMC pharmacies varied with respect to automation, procurement, drug accountability, image, participation in professional organizations, professional role, pharmaceutical care activities, technicians, and research and education. Compared with the 1992 results, the 1994 results indicated greater pharmacist involvement in patient-education activities, expanded roles for pharmacists in monitoring anticoagulation therapy and in pharmacokinetic services, and less use of pharmacists for distributive functions. In 1994, more facilities reported having an open pharmacy concept in place to encourage direct patient care initiatives. VAMCs reported greater involvement in pharmacy education in 1994 than in 1992, with more VAMCs having affiliations with pharmacy schools and clerkship and residency training programs. Responses indicated considerable variation among VAMC pharmacies in the number and types of services provided.

High level synthesis and secretion of human urokinase using a late gene promoter of the Autographa californica nuclear polyhedrosis virus.

A cDNA encoding human urokinase was inserted into the Autographa californica nuclear polyhedrosis virus (AcNPV) genome at the polyhedrin gene locus under control of a duplicated copy of the late, basic protein gene promoter. The insect-derived urokinase was produced predominantly in the form of single-chain, pro-urokinase, with a molecular mass of 50 kDa, and demonstrated fibrinolytic activity. Synthesis and secretion of urokinase was first detected at 6 hours post-infection and continued steadily throughout the infection period. Comparisons with urokinase synthesised using the very late AcNPV polyhedrin gene promoter revealed that, although the polyhedrin promoter is intrinsically stronger, the yield of secreted urokinase was higher using the basic protein gene promoter. These data support the hypothesis that the host cell secretory pathway is compromised in the very late stages of baculovirus infection and may provide an explanation for why, in general, secreted and membrane-targeted proteins are not produced to the high levels observed with other proteins, when using very late baculovirus gene promoters.

Inhibition of nitric oxide synthase--potential for a novel class of therapeutic agent?

The free-radical gas nitric oxide (NO) plays an important role in a wide and diverse range of physiological processes. As progress is made in understanding the biological function of NO, there is growing interest in the possibility that inhibitors of NO synthase (NOS) may be of clinical use in the therapy of certain disease states. The search for novel and clinically relevant inhibitors of this enzyme represents a truly multidisciplinary approach to drug screening and will no doubt benefit from the application of recombinant DNA (rDNA) technology.

The development of hemoglobin solutions as red cell substitutes.

It is clear from the trials described here that the number of different products being tested and the potential variation between batches of the same product present major problems in evaluating the safety and efficacy of hemoglobin-based oxygen carriers. The recent CBER "Points to Consider" document [42] makes clear that an understanding of the safety of oxygen carriers in humans is of paramount importance. In the event of phase II or indeed phase III trials being approved, the need may still remain for additional phase I or preclinical studies, particularly as unwanted or toxic properties of the solutions affect efficacy. It is likely that demonstrating safety and efficacy in acute hemorrhagic shock will be the most difficult task, as this is a complex clinical indication and is often accompanied by multisystem damage. The use of a hemoglobin-based oxygen carrier in this setting must have a distinct advantage over a plasma expander alone. In the application of perioperative transfusion, a decreased requirement for red cell transfusion has already been accepted as a basis for the efficacy for erythropoietin. However, in the case of a hemoglobin-based oxygen carrier, the reduction of red cell requirement in perioperative procedures would need to be balanced against any adverse drug reactions or unacceptable hemodynamic effects that may be caused by the product. It appears that there are still numerous hurdles to overcome in the development of hemoglobin-based red cell substitutes. Before these products can become established in medical practice, it is imperative that the potential mechanisms of toxicity of cell-free hemoglobin are clearly understood. Approval of hemoglobin-based oxygen carriers for clinical use will depend not only on clear demonstration of both safety and efficacy but also on risk-versus-benefit issues. Our understanding of the physiological effects of these products will evolve as progress is made in their clinical evaluation.

Haemoglobin-based red cell substitutes: current status.

Chemically modified haemoglobin solutions represent a potential alternative to the transfusion of donor blood. The theoretical advantages of these products include an oxygen delivery potential greater than that of conventional plasma expanders, prolonged shelf-life, universal compatibility and the absence of pathogenic viruses. Principal concerns have been safety issues including renal toxicity, coagulopathy and vasoactivity. The proposed indications for these solutions are primarily resuscitation of patients in haemorrhagic shock and perioperative haemodilution during elective surgery. Three products have now undergone phase I safety trials in human subjects and phase II safety and efficacy trials are planned in the near future.

Structural and functional characterisation of recombinant human haemoglobin A expressed in Saccharomyces cerevisiae.

Recombinant human HbA, produced by co-expressing alpha-globin and beta-globin chains in the yeast Saccharomyces cerevisiae, has been characterised extensively both physically and functionally. Structural studies using N-terminal sequence analysis, peptide mapping, amino acid composition analysis and electrospray MS demonstrated that the recombinant protein was identical to standard HbA purified from erythrocytes. The functional properties of the recombinant protein were assessed using equilibrium and kinetic measurements of oxygen and carbon monoxide binding. The oxygen-binding studies demonstrated that the yeast-derived HbA behaved as a fully functional, cooperative tetramer (Hill coefficient, 2.9), exhibited a normal Bohr effect and response to phosphate, and displayed a rate of oxygen dissociation identical to that of the native human molecule. The recombinant protein also showed the same characteristics of carbon monoxide combination as the standard protein. These studies demonstrate that yeast provides an ideal system for the production of Hb for structural and functional analysis and a potentially useful source of HbA for formulation into a Hb-based oxygen carrier.

Recombinant haemoglobin in the development of red-blood-cell substitutes.

Increasing concern over viral contamination of blood is spurring the development of a blood substitute which can effectively replace the oxygen-carrying capabilities of transfused erythrocytes. Solutions of chemically modified haemoglobin represent one option being evaluated for this role. More recently, recombinant-DNA techniques have enabled production of human haemoglobin in host expression systems, and progress is being made towards the creation of a genetically engineered molecule incorporating the properties required of a blood substitute.

Secretion of single-chain urokinase-type plasminogen activator from insect cells.

A cDNA encoding human urokinase-type plasminogen activator was inserted downstream from the polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus. A protein of similar Mr to urokinase (UK) was synthesized and approx. 90% was secreted from recombinant virus-infected Spodoptera frugiperda cells. Zymography and Western blotting analysis of the insect-derived protein demonstrated that it was comprised solely of the high-Mr form of UK. No low-Mr UK was detected. Amidolytic activity assays showed that 96% of the insect cell-derived UK was in the single-chain proenzyme form. The yield of UK from insect cells was 1986 international units/ml/10(6) infected cells.

Cloning and characterisation of the Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (GUT2) promoter.

The Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (GUT2) promoter and part of the protein-coding region have been isolated on a 6.3-kb genomic DNA fragment. Nucleotide sequence analysis shows that the promoter has many structural features in common with yeast glycolytic enzyme promoters. Chromosomal mapping indicates that this genomic fragment is located on chromosome XII. The GUT2 promoter has been used to construct a recombinant human albumin (reHA) secretion vector; yeast transformed with this vector secrete reHA into the culture supernatant.

A heat shock element in the phosphoglycerate kinase gene promoter of yeast.

The phosphoglycerate kinase (PGK) promoter is often employed in yeast expression vectors due to its very high efficiency. Its activity in unstressed cells has been shown to be due to an upstream activator site (UASPGK) at -402 to -479. Since levels of PGK mRNA can sometimes be elevated by heat shock of yeast cultures this investigation determined how specific deletions of PGK promoter sequences effect levels of PGK mRNA both before and after heat shock. A series of PGK promoter deletions was inserted on a high copy plasmid into cells having a TRP1 gene disruption of the solitary chromosomal PGK locus. This enabled PGK transcripts of plasmid and chromosomal origin to be distinguished by virtue of their different sizes. Certain deletions lacking UASPGK displayed activities that were very low in unstressed cells, but which increased fifty to one-hundred fold after heat shock. With UASPGK present heat shock had only a relatively small or negligible effect on PGK mRNA levels. Heat shock activation was abolished when the -256 to -377 region with homology to the heat shock element consensus of eukaryotes was deleted in addition to UASPGK, but was unaffected by the deletion of regions further downstream containing TATA- and CAAT- sequence motifs. This is the first demonstration of a heat shock element, an activator site normally found upstream of eukaryotic heat shock protein genes, as a natural constituent of a high efficiency glycolytic promoter. It is proposed that PGK may be one member of a small subset of yeast genes that are highly expressed in unstressed cells yet possess a heat shock element to ensure their continued transcription after heat shock.

The UAS of the yeast PGK gene contains functionally distinct domains.

The upstream activation site (UAS) of the yeast phosphoglycerate kinase gene (PGK) has been localised by deletion analysis (1). Here we show that the UASPGK contains two functionally distinct domains. These two domains, designated activator (A) and modulator (M), appear to be located within bases -460 to -402 and -531 to -461, respectively, relative to the initiating ATG; although it is possible that part of the M domain resides within the A domain. They have been shown, using a heterologous assay promoter, to have distinct transcriptional functions. Domain A is responsible for activation of transcription whilst domain M is required for carbon source dependent regulation of transcription. Protein-DNA binding studies have demonstrated that the DNA fragment containing domain M has high affinity for at least one specific DNA-binding protein, whilst domain A does not appear to interact strongly in protein-binding assays under the same conditions. The domain M binding activity is dependent on the carbon source in the growth medium and may be functional in the carbon source control of PGK expression.