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PubMed1989

The relationship between comprehensive functional assessment and optimal pharmacotherapy in the older patient.

Older patients hospitalized for treatment of an acute medical illness will have improved outcomes when approached in a comprehensive fashion focusing on the physical, social, and emotional aspects of life. When used by an interdisciplinary team, comprehensive functional assessment can address social, biomedical, nutrition, continence, mobility, pharmacotherapy, and psychological issues to enhance patient care. Although the appropriate use of medications is often cited as an important part of medical care for the older person, it has not been defined for this group of patients. This article outlines steps pharmacists can take to achieve optimal pharmacotherapy in older patients. Prior to attending a team conference, the pharmacist should interview the patient and review the chart. During the team conference, a comprehensive patient database will be generated that allows medications to be linked to diagnoses. To aid in selecting appropriate medications, the potential for drug-induced functional impairment of mobility, continence, and mental state is reviewed. Recommendations for therapy and establishment of therapeutic endpoints will conclude the patient conference. The pharmacist can contribute much in the process of comprehensive functional assessment and to the goal of achieving optimal pharmacotherapy in older patients.

Aged↗
PubMed1978

Experimental evolution of a new enzymatic function. II. Evolution of multiple functions for ebg enzyme in E. coli.

The evolution of ebgo enzyme of Escherichia coli, an enzyme which is unable to hydrolyze lactose, lactulose, lactobionate, or galactose-arabinoside effectively, has been directed in successive steps so that the evolved enzyme is able to hydrolyze these galactosides effectively. I show that in order for a strain of E. coli with a lacZ deletion to evolve the ability to use lactobionate as a carbon source, a series of mutations must occur in the ebg genes, and that these mutations must be selected in a particular order. The ordered series of mutations constitutes an obligatory evolutionary pathway for the acquisition of a new function for ebgo enzyme. A comparison of newly evolved strains with parental strains shows that when ebg enzyme acquires a new function, its old functions often suffer; but that in several cases old functions are either unaffected or are improved. I conclude that divergence of functions catalyzed by an enzyme need not require gene duplication.

Biological Evolution↗
PubMed1981

An electrical tuning mechanism in turtle cochlear hair cells.

1. Intracellular recordings were made from single cochlear hair cells in the isolated half-head of the turtle. The electrical responses of the cells were recorded under two conditions: (a) when the ear was stimulated with low-intensity tones of different frequencies and (b) when current steps were injected through the intracellular electrode. The aim of the experiments was to evaluate the extent to which the cochlea's frequency selectivity could be accounted for by the electrical properties of the hair cells.2. At low levels of acoustic stimulation, the amplitude of the hair cell's receptor potential was proportional to sound pressure. The linear tuning curve, which is defined as the sensitivity of the cell as a function of frequency when the cell is operating in its linear range, was measured for a number of hair cells with characteristic frequencies from 86 Hz to 425 Hz.3. A rectangular current passed into a hair cell elicited a membrane potential change consisting of a damped oscillation superimposed on a step. Small currents produced symmetrical oscillations at the beginning and end of the pulse. Larger currents increased the initial ringing frequency if depolarizing and decreased it if hyperpolarizing.4. For small currents the frequency of the oscillations and the quality factor (Q) of the electrical resonance derived from the decay of the oscillations were close to the characteristic frequency and Q of the hair-cell linear tuning curve obtained from sound presentations.5. The hair cell's membrane potential change to small-current pulses or low-intensity tone bursts could be largely described by representing the hair cell as a simple electrical resonator consisting of an inductance, resistor and capacitor.6. When step displacements of 29-250 nm were applied to a micropipette, placed just outside a hair cell in the basilar papilla, an initial periodic firing of impulses could be recorded from single fibres in the auditory nerve. Currents of up to 1 nA, injected through the same micropipette, failed to produce any change in the auditory nerve discharge. The experiment demonstrates that current injection does not produce gross movements of the electrode tip.7. The contribution of the electrical resonance to hair-cell tuning was assessed by dividing the linear tuning curve by the cell's impedance as a function of frequency. The procedure assumes that the electrical resonance is independent of other filtering stages, and on this assumption the resonance can account for the tip of the acoustical tuning curve.8. The residual filter produced by the division was broad; it exhibited a high-frequency roll-off with a corner frequency at 500-600 Hz, similar in all cells, and a low-frequency roll-off, with a corner frequency from 30 to 350 Hz which varied from cell to cell but was uncorrelated with the characteristic frequency of the cell.9. The phase of the receptor potential relative to the sound pressure at the tympanum was measured in ten cells. For low intensities the phase characteristic was independent of the sound pressure. At low frequencies the receptor potential led the sound by 270-360 degrees , and in the region of the characteristic frequency there was an abrupt phase lag of 90-180 degrees ; the abruptness of the phase change depended upon the Q of the cell.10. The calculated phase shift of the electrical resonator as a function of frequency was subtracted from the phase characteristic of the receptor potential. The subtraction removed the sharp phase transition around the characteristic frequency, and in this frequency region the residual phase after subtraction was approximately constant at +180 degrees . This is consistent with the idea that the hair cells depolarize in response to displacements of the basilar membrane towards the scala vestibuli. The high-frequency region of the residual phase characteristic was similar in all cells.11. It is concluded that each hair cell contains its own electrical resonance mechanism which accounts for most of the frequency selectivity of the receptor potential. All cells also show evidence of a broad band-pass filter, the high frequency portion of which may be produced by the action of the middle ear.

Animals↗
PubMed1987

Measurement of postcryopreservation viability.

For any tissue, there is a cell viability threshold below which the ability of the tissue to maintain itself and function will eventually be compromised. During cryopreservation and subsequent thawing of tissues there are many steps involved, each with attendant potential risks for reduction of viability. To determine the effectiveness of any cryopreservation procedure it is important to select appropriate assays. In this manuscript viability assays, in general, are reviewed from a biological viewpoint prior to review of methods employed for assessment of heart valve viability. Both in situ and in vitro assays of heart valve viability indicate that valve mechanical properties and the majority of fibroblasts, which are responsible for maintenance of the valve connective tissue, are retained after cryopreservation.

Cryopreservation↗
PubMed1994

Quinolone resistance mutations in the DNA gyrase gyrA and gyrB genes of Staphylococcus aureus.

A 6.4-kb DNA fragment containing the DNA gyrase gyrA and gyrB genes was cloned and sequenced from the quinolone-susceptible Staphylococcus aureus type strain ATCC 12600. An expression plasmid was constructed by inserting the cloned genes into the Escherichia coli-S. aureus shuttle vector pAT19, and deletion plasmids carrying only functional gyrA and gyrB genes were derived from this plasmid. An efficient transformation system for S. aureus RN4220 was established by using these plasmids. Quinolone-resistant mutants of S. aureus RN4220 were isolated by three-step selection with quinolones. The first- and second-step mutants were considered to be transport mutants, and the third-step mutants were divided into five groups with respect to their resistance patterns and transformation results with gyrA and gyrB genes. Sequencing analysis of the resulting mutant gyrase genes showed that they had the following point mutations: group 1, Ser-84 (TCA) to Leu (TTA) in GyrA; group 2, Ser-84 (TCA) to Ala (GCA), Ser-85 (TCT) to Pro (CCT), or Glu-88 (GAA) to Lys (AAA) in GyrA; group 3, Asp-437 (GAC) to Asn (AAC) in GyrB; group 4, Arg-458 (CGA) to Gln (CAA) in GyrB; and group 5, Ser-85 (TCT) to Pro (CCT) in GyrA and Asp-437 (GAC) to Asn (AAC) in GyrB. When the gyrA and/or gyrB mutants were transformed with the wild-type gyrA and/or gyrB plasmids, they became quinolone susceptible, but transformants with the plasmids having the same mutations on the gyrA and/or gyrB genes did not confer susceptibility. These results indicate that mutations in both gyrA and gyrB can be responsible for quinolone resistance in S. aureus.

Amino Acid Sequence↗
PubMed1988

Isolation and analysis of hepatoma nuclear proteins using monoclonal antibodies.

Monoclonal antibodies were prepared to nuclear nonhistone proteins from a 2-aminoacetyl fluorine-induced transplantable rat hepatocellular carcinoma. These antibodies recognized a total of six distinct antigens as revealed by molecular weight analysis. Studies of antigen specificity with respect to various tissues, tumors, cultured cells, and oncodevelopmental stages indicated that these nuclear species could be divided into two categories. Four antigens were classified as tumor related since they were significantly enriched in tumor tissue as compared to tissues of the normal adult rat. The remaining two antigens were detected only in tumors and transformed cells; one, only in certain hepatomas. Thus, these antigens were classified as tumor specific. As an initial step toward elucidating the function of these proteins, each antigen was isolated by immunoaffinity chromatography, radioiodinated in situ, and analyzed for the ability to bind DNA. Three antigens were positive for DNA binding, and one of these was selectively released from tumor nuclei with the transcriptionally active chromatin upon digestion with micrococcal nuclease. The implications of these results for the possible functional contribution of the six tumor antigens to transformation is discussed.

2-Acetylaminofluorene↗
PubMedDate not supplied

[Thiamine diphosphate level and metabolism in allergic myocarditis and treatment with peloid].

Enzymatic systems involved in thiamin metabolism were studied in experimental allergic myocarditis. Development of inflammation in myocardium was accompanied by a distinct activation of thiamin pyrophosphatase which catalyzed the most important step responsible for deterioration of the coenzyme functions. The stabilizing effect of peloid on the intracellular pool and compartmentalization of thiamin in myocarditis involved equilibration of the anabolic and catabolic reactions in the coenzyme metabolism, mainly due to selective inhibition of the thiamin pyrophosphatase activity stimulated under these conditions.

Animals↗
PubMed1994

Therapy for hepatitis B virus infection.

Major advances have been made in the therapy of chronic viral hepatitis B during the past several years. This period has witnessed the publication of large, multicenter trials of recombinant interferon alfa for chronic hepatitis B in the United States and the completion of several similarly designed studies in North America, Europe and Asia. These studies have defined an initial response rate of approximately 40% to 50%. In contrast to the experience with chronic hepatitis C, loss of viral replication is generally sustained. Repeat courses of therapy are likely to result in the same type of response as that observed initially. Quantitative assessment of viral replication (HBV DNA, HBeAg) is important in predicting the likelihood of response and in monitoring patients during therapy. Perhaps the most compelling reason to treat chronic hepatitis B with interferon is the disappearance of circulating HBsAg in one third of responders with a further increase in frequency of this phenomenon as follow-up continues. Another important advantage to treatment is the striking degree of histologic improvement that is frequently observed years after a response has been achieved. Although a substantial number of patients do not respond to interferon, several promising agents that should allow for a greater degree of success, when used either alone or in combination with interferon, are under study. A short course of corticosteroids prior to interferon appears to improve response rates in patients who have low ALT levels at baseline and has been the preferred approach for these patients at our medical center. Progress is being made in the development of safer interferon regimens for patients with mild-to-moderate hepatic decompensation. Nonetheless, even patients with marginal synthetic function, as reflected by albumin levels within the low-normal range, appear to be at greater risk for complications during therapy and should preferably be referred for inclusion in research programs. Appropriate patient selection remains a critical step for maximizing the safety as well as efficacy of interferon treatment.

Adrenal Cortex Hormones↗
PubMed1992

(H+,K+)-ATPase inhibiting 2-[(2-pyridylmethyl)sulfinyl]benzimidazoles. 4. A novel series of dimethoxypyridyl-substituted inhibitors with enhanced selectivity. The selection of pantoprazole as a clinical candidate.

[(Pyridylmethyl)sulfinyl]benzimidazoles 1 (PSBs) are a class of highly potent antisecretory (H+,K+)-ATPase inhibitors which need to be activated by acid to form their active principle, the cyclic sulfenamide 4. Selective inhibitors of the (H+,K+)-ATPase in vivo give rise to the nonselective thiophile 4 solely at low pH, thus avoiding interaction with other thiol groups in the body. The propensity to undergo the acid-catalyzed transformation is dependent on the nucleophilic/electrophilic properties of the functional groups involved in the formation of 2 since this step is both rate-determining and pH-dependent. The aim of this study was to identify compounds with high (H+,K+)-ATPase inhibitory activity in stimulated gastric glands possessing acidic pH, but low reactivity (high chemical stability) at neutral pH as reflected by in vitro (Na+,K+)-ATPase inhibitory activity. The critical influence of substituents flanking the pyridine 4-methoxy substituent present in all derivatives was carefully studied. The introduction of a 3-methoxy group gave inhibitors possessing a combination of high potency, similar to omeprazole and lansoprazole, but increased stability. As a result of these studies, compound 1a (INN pantoprazole) was selected as a candidate drug and is currently undergoing phase III clinical studies.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1992

A systematic search for protein signature sequences.

Signature sequences are contiguous patterns of amino acids 10-50 residues long that are associated with a particular structure or function in proteins. These may be of three types (by our nomenclature): superfamily signatures, remnant homologies, and motifs. We have performed a systematic search through a database of protein sequences to automatically and preferentially find remnant homologies and motifs. This was accomplished in three steps: 1. We generated a nonredundant sequence database. 2. We used BLAST3 (Altschul and Lipman, Proc. Natl. Acad. Sci. U.S.A. 87:5509-5513, 1990) to generate local pairwise and triplet sequence alignments for every protein in the database vs. every other. 3. We selected "interesting" alignments and grouped them into clusters. We find that most of the clusters contain segments from proteins which share a common structure or function. Many of them correspond to signatures previously noted in the literature. We discuss three previously recognized motifs in detail (FAD/NAD-binding, ATP/GTP-binding, and cytochrome b5-like domains) to demonstrate how the alignments generated by our procedure are consistent with previous work and make structural and functional sense. We also discuss two signatures (for N-acetyltransferases and glycerol-phosphate binding) which to our knowledge have not been previously recognized.

Amino Acid Sequence↗
PubMed1995

The molecular biology of multidomain proteins. Selected examples.

The aim of this review is to give an overview of the contribution molecular biology can make to an understanding of the functions and interactions within multidomain proteins. The contemporary advantages ascribed to multidomain proteins include (a) the potential for metabolite channelling and the protection of unstable intermediates; (b) the potential for interactions between domains catalysing sequential steps in a metabolic pathway, thereby giving the potential for allosteric interactions; and (c) the facility to produce enzymic activities in a fixed stoichiometric ratio. The alleged advantages in (a) and (b) however apply equally well to multi-enzyme complexes; therefore, specific examples of these phenomena are examined in multidomain proteins to determine whether the proposed advantages are apparent. Some transcription-regulating proteins active in the control of metabolic pathways are composed of multiple domains and their control is exerted and modulated at the molecular level by protein-DNA, protein-protein and protein-metabolite interactions. These complex recognition events place strong constraints upon the proteins involved, requiring the recognition of and interaction with different classes of cellular metabolites and macromolecules. Specific examples of transcription-regulating proteins are examined to probe how their multidomain nature facilitates a general solution to the problem of multiple recognition events. A general unifying theme that emerges from these case studies is that a basic unitary design of modules provided by enzymes is exploited to produce multidomain proteins by a complex series of gene duplication and fusion events. Successful modules provided by enzymes are co-opted to new function by selection apparently acting upon duplicated copies of the genes encoding the enzymes. In multidomain transcription-regulating proteins, former enzyme modules can be recruited as molecular sensors that facilitate presumed allosteric interactions necessary for the molecular control of transcription.

Animals↗
PubMed1981

Role of blood platelets and prostaglandins in coronary artery disease.

In the last decade, several studies evaluating blood platelet function in patients with coronary heart disease have been reported. Although several platelet function abnormalities such as enhanced platelet aggregation, decreased platelet survival and increase in platelet release reaction in the stable condition and during stress in patients with myocardial ischemia have been recognized, the mechanism of these abnormalities is just beginning to be understood. Discovery of certain platelet and endothelium-generated prostaglandins has provided some information as to the possible mechanism of platelet dysfunction. Abnormalities of prostaglandin production and platelet sensitivity to various prostaglandins may have an important bearing on the enhanced platelet aggregation in vivo, genesis of atherosclerosis and probably precipitation of acute ischemic events. Since the discovery of these prostaglandins, the precise mode of action of several commonly used platelet-active drugs has been clarified. Development of new drugs acting at selective steps in the prostaglandin pathways may provide some exciting novel therapeutic procedures in patients with coronary heart disease.

Acute Disease↗
PubMed1985

Genetic analysis of spontaneous resistance to ampicillin in Neisseria gonorrhoeae.

Step-wise intrinsic resistance to ampicillin in Neisseria gonorrhoeae was analyzed genetically by DNA-mediated transformation experiments. A first-step ampicillin-resistant (Ampr1) mutant and a second-step ampicillin-resistant (Ampr2) mutant generated during sequential selection were used in these studies. Each selection step was accompanied by an approximate twofold increase in resistance. Four amp alleles were found to account for full resistance of the Ampr2 phenotype. All four amp alleles lie among a cluster of genes which code for ribosomal functions. This region has the map order rif str fus tet cam. First-step resistance was caused by two amp alleles, ampA2 and ampB1, neither of which independently caused detectable ampicillin resistance. Outcrossing of the ampA2 or the ampB1 mutation resulted in wild-type susceptibility to ampicillin. Mapping studies indicate that ampB1 lies between str and fus, whereas ampA2 lies to the right of cam. Second-step resistance required two mutations, ampC3 and ampD4, in addition to ampB1 and ampA2. Transformation of ampC3 to ampC3+ in an Ampr2 mutant resulted in the Ampr1 phenotype. Both ampC3 and ampD4 showed transformation linkage to rif and str. ampC3 was positioned at a site between rif and str. ampD4 apparently occupied a site, outside of the rif-str region, proximal to rif and distal to str. We postulate the gene order to be ampD rif ampC str ampB fus tet cam ampA.

Alleles↗
PubMed1984

Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step.

Asparagine-linked glycosylation is a form of covalent modification that distinguishes proteins that are either membrane bound or are in cellular compartments topologically outside of the cell from those proteins that remain soluble in the cytoplasm. This type of glycosylation occurs stepwise, with core oligosaccharide added in the endoplasmic reticulum and subsequent modifications occurring in the golgi. We used tunicamycin, an inhibitor of one of the earliest steps in the synthesis of N-linked oligosaccharide, to select for mutants that are resistant to this antibiotic. Genetic, biochemical, and physiological experiments led to the following conclusions. The synthesis of N-linked oligosaccharide is an essential function in cells. In contrast to mammalian cells, yeast cells do not transport tunicamycin by a glucosamine transport function. We identified a gene, ALG7, that is probably the structural gene for UDP-N-acetylglucosamine-1-P transferase, the enzyme inhibited by tunicamycin. Dominant mutations in this gene result in increased activity of the transferase and loss of the ability of the cell to sporulate. In addition, we identified another gene, TUN1, in which recessive mutations result in resistance to tunicamycin. The ALG7 and TUN1 genes both map on chromosome VII.

Asparagine↗
PubMedDate not supplied

Cytoskeleton and epithelial polarity.

The membrane surface of polarized epithelial cells can be divided in apical and basolateral domains that differ in molecular composition and function. Components of the cytoskeleton are involved in critical steps of both generation and maintenance of cell polarity. Generation of polarity is controlled by microtubules that serve as uniformly aligned and polarized cytoplasmic guiding structures for the vectorial and selective transport of Golgi-derived carrier vesicles to the apical cell surface. Targeting of membrane proteins to the basolateral cell surface does not depend on microtubules but follows the constitutive bulk flow of membranes. Once inserted into the lipid bilayer several membrane proteins such as the kidney anion exchanger 1 (AE1) and the sodium pump become immobilized at specialized microdomains of the lateral cell surface. Evidence is provided that both membrane proteins are linked via ankyrin to the spectrin-based membrane cytoskeleton that underlies the basolateral membrane domain. Linkage of these and other integral membrane proteins to the cytoskeleton may not only place them to specialized sites of the plasma membrane but may also prevent these transporters from clustering and endocytosis, thus helping them to stay at the cell surface. In search of sequence motifs involved in binding of integral membrane proteins to components of the cytoskeleton we found that the binding interface of AE1 to protein 4.1 (an actin and spectrin cross-linking protein) consists of a cluster of five amino acid residues, namely IRRRY in AE1 and LEEDY on protein 4.1. This motif may play a more general role in cytoskeleton membrane linkages.

Amino Acid Sequence↗
PubMed1985

[Current in vivo and in vitro diagnosis of thyroid diseases].

A single method which is sufficient to meet all diagnostic requirements in patients with thyroid disorders does not exist. In selecting a test combination one should always consider, that differentiation of patients with hyper- or hypothyroidism from those with euthyroidism should involve the least effort. A meaningful step-by-step diagnostic work-up should always start with individually selected in vitro tests, followed by in vivo methods such as ultrasonography, radionuclide scanning and X-ray examination. This accords with the recommendations for diagnosis of disturbed thyroid function and thyroid disorders published recently by the thyroid section of the German Association of Endocrinology.

Humans↗
PubMed1985

Characterization of a Euglena gracilis chloroplast RNA polymerase specific for ribosomal RNA genes.

Euglena gracilis chloroplasts contain a 145,000-base pair chromosome that encodes genes for ribosomal, transfer, and messenger RNAs. These genes are transcribed within the organelle by chloroplast RNA polymerase activities that are specific for different classes of RNA. Two transcriptional activities have been isolated from Euglena chloroplasts. (Greenberg, B. M., Narita, J. O., DeLuca-Flaherty, C., Gruissem, W., Rushlow, K. A., and Hallick, R. B. (1984) J. Biol. Chem. 259, 14880-14887). One, the "soluble extract," contains enzymes active in tRNA transcription and processing. The other activity, the transcriptionally active chromosome, consisting of a chloroplast DNA-dependent RNA polymerase tightly bound to chloroplast DNA, only transcribes rRNA genes even though the entire chloroplast genome is present. We have extensively purified the transcriptionally active chromosome using high salt concentrations to dissociate loosely bound proteins. The result is a highly enriched extract containing three major polypeptides of Mr 116,000-118,000, 83,000-88,000, and 24,000-26,000 that retains complete selectivity for rDNA transcription. It is probable that one, or both, of the high molecular weight proteins are functional components of the DNA-dependent RNA polymerase. The identification and characterization of the transcriptionally active chromosome is a first step towards understanding how chloroplast rRNA synthesis is regulated.

Chloroplasts↗
PubMedDate not supplied

Modulation of human immune responsiveness in vitro by auranofin.

The effect of auranofin (AF) on in vitro correlates of human immune responsiveness was examined. AF inhibited mitogen induced human lymphocyte proliferation and the generation of immunoglobulin secreting cells in a concentration dependent manner. The inhibition was most effective when AF was present from the initiation of culture indicating that this drug blocked a critical early step in lymphocyte activation. Marked inhibition of mitogen responsiveness was observed as a result of a 1-h preincubation with AF. The brief preincubation with low concentrations of AF (0.3 micrograms/ml) resulted in a selective inhibition of the accessory function of monocytes but had no effect on potential lymphocyte responsiveness. Preincubation with higher concentrations of AF (greater than 0.6 micrograms/ml) resulted in a more non-specific inhibition of both monocyte and lymphocyte function. These data support the conclusion that AF may function as an immunosuppressive agent.

Antibody Formation↗