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PubMed1992

Hepatocyte heterogeneity in bile formation and hepatobiliary transport of drugs.

In the past two decades many studies have been devoted to the involvement of the periportal (zone-1) and perivenous (zone-3) hepatocytes in bile formation and hepatobiliary transport of endogenous and exogenous compounds. It became clear that such a heterogeneity in transport function can, in principle, be due to the different localization of the cells in the acinus with respect to the incoming blood, to intrinsic differences between the cells or to both. In this review we first discuss the techniques used to study hepatocyte heterogeneity in hepatobiliary transport function. Combinations of such techniques can be used to discriminate between cellular heterogeneity due to acinar localization as opposed to intrinsic differences. These techniques include: normal and retrograde perfusions of isolated perfused livers; autoradiographic, fluorimetric and histochemical localization of injected substrates; separation of isolated hepatocytes into fractions enriched in periportal and perivenous cells; measurements of fluorescent surface signals with microlight guides; selective zonal toxicity, and pharmacokinetic modelling and analysis. Subsequently, for each of the rate-limiting steps in the hepatobiliary transport of organic compounds, the basic mechanisms are summarized and the available knowledge on the involvement of the cells from the various zones in these transport steps is discussed. The available literature data indicate that heterogeneity in transport function is often due to the localization of the cells in the acinus: the periportal cells are the first to come into contact with the portal blood and are thus exposed to the highest substrate concentration. Consequently they obtain the most prominent task in further disposition of the particular compound. It follows that the extent of involvement of the perivenous cells in drug disposition is implicitly determined by the activity of the periportal cells. Because of the potential saturation of elimination processes in the periportal cells, the involvement of perivenous cells may vary with the input concentration. In addition, real intrinsic differences have been established in the hepatobiliary transport of some substrates. These are probably based on differences in the cellular content of carrier- and receptor-binding and/or metabolizing proteins. In some cases these intrinsic differences may be secondary to existing sinusoidal gradients of endogenous compounds, such as O2, amino acids, bile acids or monosaccharides. Yet, data on the heterogeneity of hepatocytes in the various transport steps are far from complete or are even totally lacking, especially for human liver. A multi-experimental approach and advanced technology will be needed in the future to gain more insight into the acinar organization of bile formation and hepatobiliary transport of drugs in the human.

Animals↗

PubMed1989

Role of protein X in the function of the mammalian pyruvate dehydrogenase complex.

Two lipoyl-bearing subunits--the dihydrolipoyl transacetylase and protein X--form the core of the mammalian pyruvate dehydrogenase complex. Selective removal of the lipoyl domain of protein X results in loss in the activity of the complex with a relationship suggesting the involvement of the lipoyl domain of protein X in a key but not rate limiting step. The dihydrolipoyl dehydrogenase component markedly reduces both the cleavage of protein X and the loss in activity. Using a microplate binding assay, we demonstrate that the lipoyl domain of protein X and the transacetylase component contribute to the binding of the dihydrolipoyl dehydrogenase component. These roles of protein X in the catalytic function and organization of the complex require new reactions and afford an explanation for the unusual stoichiometry of dihydrolipoyl dehydrogenase dimers in the complex.

Animals↗

PubMed1990

Frameshift autoregulation in the gene for Escherichia coli release factor 2: partly functional mutants result in frameshift enhancement.

The regulation of release factor 2 (RF-2) synthesis in Escherichia coli occurs, at least in part, through autoregulatory feedback exerted at a unique frameshifting step required during RF-2 translation. We have constructed fusions between the genes for RF-2 and E. coli trpE which make direct measurement of frameshifting efficiency possible since both products of regulation, the termination product and the frameshift product, are stable. The addition of purified RF-2 to in vitro expressions of these fusion genes was found to result in decreased frameshifting and increased termination at the regulation site. The frame-shifted trpE-RF-2 products synthesized from these fusions are unique with respect to their functional release factor activities; when tested in assays of two intermediate steps of translational termination, they were found to be partially active for the function of ribosome binding, but inactive for peptidyl-tRNA hydrolysis (release). These are the first examples of release factor mutants selectively active for only one of these function. In vivo these chimeric proteins promote large increases in frameshifting at the RF-2 frameshift region, thereby reversing normal negative autoregulatory feedback and instead supporting fully efficient frameshifting in their own synthesis. This activity provides new evidence for the importance of ribosomal pausing in directing efficient frameshifting at the RF-2 frameshift region.

Base Sequence↗

PubMed1988

[ACE inhibitors in the diagnosis and therapy of various forms of hypertension].

The diagnostic and therapeutic aspects of ACE inhibitors in patients with various forms of hypertension are discussed. The immediate blood pressure reduction after administration of an ACE inhibitor is significantly more marked in patients with renovascular than with essential hypertension. However, analysis of the individual blood pressure response after captopril does not allow a clear distinction between these two forms of hypertension. In patients with moderate hypertension the reduction of blood pressure with ACE inhibitors is similar to that with betablockers. Their use as a first step agent in moderate hypertension, however, is limited to selected patients. In severe drug-resistant hypertension treatment with ACE inhibitors is well established. Cases with renovascular hypertension may suffer a deterioration of renal function under ACE inhibitors. Other side effects are uncommon where renal function is normal or where the dose is adapted to the decreased glomerular filtration rate.

Adrenergic beta-Antagonists↗

PubMed1986

Characterization of human mononuclear cells after positive selection with immunomagnetic particles.

We have investigated the possibility to employing magnetic monodisperse polymer particles for positive selection of human peripheral blood mononuclear cell populations. By carefully titrating the ratio between particles and cells we succeeded in isolating a number of cell populations that could be cultivated subsequently in vitro for functional studies. The success of the procedure is partly dependent on the properties of the monoclonal antibodies used to sensitize the cells. Provided these antibodies do not react with membrane structures involved in the transduction of activating signals, highly purified, quiescent cell populations can be recovered in a single fractionation step. In most instances particles will detach from the isolated cells by overnight culture, and the particles can then be removed from the system by a suitable magnet. T lymphocytes, subpopulations of T lymphocytes, and B lymphocytes have been isolated in this way and studied in a variety of functional assay systems. Comparison with cells obtained after negative selection clearly demonstrates the usefulness of this technique, especially if the membrane marker selected for it is not directly engaged in the activation processes.

Antibodies, Anti-Idiotypic↗

PubMed1988

Generation of high specificity of effect through low-specificity binding of proteins to DNA.

It is proposed that proteins can bind with relatively low-affinity and specificity to multiple sites, defined as sequence motifs, on polynucleotide chains, and that such binding can collectively be turned into high-affinity, high-specificity binding through cooperative effects, especially when the sequence motifs recur periodically. The selection of individual nucleotides has in general been thought to be the condition of the existence and conservation of function in most of the noncoding sequences. This condition seems unnecessary. Calculations are presented as a step in the direction of giving credibility to a model of stable gene repression.

DNA↗

PubMed1975

Studies on the nature and function of polygenic loci in Drosophila. III. Veinlet modifiers having region-specific effects upon the vein pattern.

Polygenic modifiers affecting the expression of the mutant veinlet were studied to determine whether each acts specifically upon one vein or wing region or whether they affect the venation pattern in some general way. Selection experiments showed that the L4 vein can be modified independently of the L2 and L3 veins. Similarly, the L2 vein can be shortened while the L3 is selected to be longer, although there is some interdependence between the L2 and L3 veins. Assays of heterozygous whole chromosome effects show that different chromosomes are involved in the responses of separate veins, and one polygenic locus causing a decrease in L4 vein length has been isolated. Substitutions of whole chromosomes from selection lines into unselected backgrounds of non-homologous mutants demonstrate that the selected modifiers have the same qualitative effects upon other mutants having similar phenotypes. These results support the hypothesis that the polygenic modifiers affecting veinlet expression function independently of the veinlet locus, presumably by influencing common steps in the developmental processes leading to the formation of individual veins.

Alleles↗

PubMed1986

Functional channel formation associated with cytotoxic T-cell granules.

Lymphocyte granules from cytotoxic T-lymphocyte lines A2, A11, and R8 were enriched by subcellular fractionation using a Percoll gradient. Granule-enriched fractions showed potent hemolytic activity in the presence of Ca2+. Isolated granules induced rapid Ca2+-dependent membrane depolarization of J774 macrophage-like cells. When tested in planar bilayers, granules induced the formation of Ca2+-dependent functional ion channels of large conductance steps of 1-6 nS in 0.1 M NaCl. Granule-induced channels were resistant to closing by an increase in transmembrane potential, with few channels shifting to the closed state only at voltages of greater than 70 mV, following a Poisson process. These channels showed poor ion selectivity and were permeable to all monovalent and divalent ions (K+, Na+, Li+, Cl-, Ca2+, Mg2+, Zn2+, Ba2+). Ultrastructural examination of soluble granule proteins incubated for 48 hr at 37 degrees C in the presence of Ca2+ revealed ring-like structures of 150-200 A. Structural and functional channel formation may be involved in cytolysis induced by cytotoxic T lymphocytes.

Animals↗

PubMed1992

A critical evaluation of the magnetic cell sorter and its use in the positive and negative selection of CD45RO+ cells.

In this paper, we report on our year-long experience with the magnetic cell sorter (MACS), and present a critical evaluation of its pitfalls and benefits. Satisfactory separation of lymphocytes into subsets with preservation of function can be achieved, but there are several drawbacks: in comparison with Dynal beads, MACS results in a higher cell loss due to the increased number of separation steps and requires depletion of plastic-adherent cells as these will engulf microbeads and contaminate the enriched fraction, and is more expensive. The advantage of MACS over Dynal beads is that the microbeads are biodegradable and do not interfere with proliferation assays: both the depleted and enriched fractions of cells can therefore be used in culture immediately following separation. We used MACS for the positive and negative selection of CD45RO cells: the enriched fraction was of high purity (greater than 98%), but a depleted fraction of greater than 90% purity could not be obtained even after running the same sample over three separating columns. Dynabeads, on the other hand, achieved 98% pure CD45RO-depleted fractions after three separation runs.

Antigens, CD↗

PubMed1991

Normal development and function of CD8+ cells but markedly decreased helper cell activity in mice lacking CD4.

T cells express T-cell antigen receptors (TCR) for the recognition of antigen in conjunction with the products of the major histocompatibility complex. They also express two key surface coreceptors, CD4 and CD8, which are involved in the interaction with their ligands. As CD4 is expressed on the early haemopoietic progenitor as well as the early thymic precursor cells, a role for CD4 in haemopoiesis and T-cell development is implicated. Thymocytes undergo a series of differentiation and selection steps to become mature CD4+8- or CD4-8+ (single positive) T cells. Studies of the role of CD4+ T cells in vivo have been based on adoptive transfer of selected or depleted lymphocytes, or in vivo treatment of thymectomized mice with monoclonal antibodies causing depletion of CD4+ T cells. In order to study the role of the CD4 molecule in the development and function of lymphocytes, we have disrupted the CD4 gene in embryonic stem cells by homologous recombination. Germ-line transmission of the mutation produces mutant mouse strains that do not express CD4 on the cell surface. In these mice, the development of CD8+ T cells and myeloid components is unaltered, indicating that expression of CD4 on progenitor cells and CD4+ CD8+ (double positive) thymocytes is not obligatory. Here we report that these mice have markedly decreased helper cell activity for antibody responses, although cytotoxic T-cell activity against viruses is in the normal range. This differential requirement for CD4+ helper T cells is important to our understanding of immune disorders including AIDS, in which CD4+ cells are reduced or absent.

Animals↗

PubMed1990

Exon mutations uncouple 5' splice site selection from U1 snRNA pairing.

It has previously been shown that a mutation of yeast 5' splice junctions at position 5 (GUAUGU) causes aberrant pre-mRNA cleavages near the correct 5' splice site. We show here that the addition of exon mutations to an aberrant cleavage site region transforms it into a functional 5' splice site both in vivo and in vitro. The aberrant mRNAs are translated in vivo. The results suggest that the highly conserved G at the 5' end of introns is necessary for the second step of splicing. Further analyses indicate that the location of the U1 snRNA-pre-mRNA pairing is not affected by the exon mutations and that the precise 5' splice site is selected independent of this pairing.

Base Sequence↗

PubMed1990

Deriving criteria for hearing impairment in the elderly: a functional approach.

We describe a method for deriving criteria for hearing impairment in the elderly based on self-reported handicap. Using the Sickness Impact Profile (SIP) and Hearing Handicap Inventory for the Elderly - Screening (HHIE-S) version as functional measures of handicap, the analysis proceeded in five steps: 1. Audiometric thresholds at various frequencies were inter-correlated. This was done both within and between ears. 2. Better and poorer ear thresholds were determined for each frequency, and these were correlated with the HHIE-S and SIP scores. 3. Using the HHIE-S and SIP scores as dependent variables, stepwise multiple linear regressions were used to select the frequencies that explained the most variance in the functional scales. 4. Using the HHIE-S and SIP as standards, receiver operating curves were constructed for each frequency to select the threshold level that provided the best test accuracy. 5. The newly-derived criteria were then compared against four other "traditional" criteria of hearing impairment. In general, the newly-derived criteria combined a relatively low frequency with a relatively high frequency, with the low frequency being functionally more important. Depending on the functional scale used, the threshold level was in the 25 to 35-dB range for the lower frequencies and 40 to 45-dB for the higher frequencies. These features provide a suitable compromise to the current debate over which threshold levels comprise the best discrimination of aged persons who are hearing-impaired. Future research should focus on developing consensus standards for functional hearing impairment and handicap in the elderly.

Age Factors↗

PubMed1987

Formation and regulation of platelet and fibrin hemostatic plug.

Formation of a hemostatic plug represents one of the earliest responses to vessel wall injury. Platelets react to any discontinuity in the vascular endothelium through initial contact, spreading, and formation of a thrombus (or aggregate). This development of a primary hemostatic plug requires platelet membrane receptors through which the adhesive macromolecules, von Willebrand factor (vWF) and fibrinogen, anchor platelets to the vessel wall and link them to each other. There are two receptor pathways--classic and alternative--for the binding of vWF to platelets; the latter induced by thrombin, and adenosine diphosphate (ADP) is shared with fibrinogen. Synthetic peptides, patterned after known binding domains of adhesive molecules, have been designed to inhibit their interactions with platelet receptors. A secondary hemostatic plug, composed of platelets enmeshed in fibrin, results from the action of thrombin, which is not only essential for formation of fibrin but also for exposure of platelet receptors for adhesive molecules and for "activation" of factors V and VIII. Thrombin generation is greatly enhanced through the activity of the prothrombinase complex formed on the surface of platelets, perturbed endothelial cells, and leukocytes. A pivotal event is activation of factor X through the intrinsic and extrinsic coagulation pathways. Binding of factors IXa and VIIa to the vascular endothelium represents a localized mechanism for factor Xa generation. Formation of a platelet and fibrin thrombus is controlled by regulatory mechanism: prostacyclin, endogenous heparin-antithrombin III complex, thrombomodulin-protein C-protein S system, and the fibrinolytic system. The balance of all components--vessel wall, platelets, adhesive and coagulation proteins, regulatory mechanisms--determines the effectiveness of the hemostatic plug in maintaining the structural and functional integrity of the circulatory system. An approach to detection of hemostatic derangements in patients at risk evolves from a full understanding of inherited and acquired deficiencies affecting each step of hemostatic plug formation and from selective use of laboratory tests.

Blood Coagulation Tests↗

PubMed1989

Using genetically-defined rodent strains for the identification of hippocampal traits relevant for two-way avoidance behavior: a non-invasive approach.

Genetically-defined rodent strains permit the identification of hippocampal traits which are of functional relevance for the performance of two-way avoidance behavior. This is exemplified here by analyzing the relationship between infrapyramidal mossy fibers (a tiny projection terminating upon the basal dendrites of hippocampal pyramidal neurons) and two-way avoidance learning in about 800 animals. The necessary steps include 1) identification of structural traits sensitive to selective breeding for extremes in two-way avoidance, 2) testing the robustness of the associations found by studying individual and genetical correlations between hippocampal traits and behavior, 3) establishing causal relationships by Mendelian crossing of strains with extreme structural traits and studying the behavioral consequences of such structural 'randomization', 4) confirming causal relationships by manipulating the structural variable in inbred (isogenic) strains, thereby eliminating the possibility of genetic linkage, and 5) ruling out the possibility of spurious associations by studying the correlations between the hippocampal trait and other behaviors known to depend on hippocampal functioning. In comparison with the classical lesion approach for identifying relationships between brain and behavior, the present procedure appears to be superior in two aspects: it is non-invasive, and it focuses automatically on those brain traits which are used by natural selection to shape behaviorally-defined animal populations, i.e., it reveals the natural regulators of behavior.

Animals↗

PubMed1989

Post-translational modifications of proteins: some problems left to solve.

Three major questions regarding the post-translational modification of amino acid side chains in proteins are briefly considered: (1) What are the biological functions of the reactions, (2) what is the specificity of the processing reactions in selecting only a few or sometimes even only one residue for modification, and (3) how do we solve the uniqueness of the processing steps in the production of recombinant proteins? The answers to these questions are not obvious at this time.

Protein Processing, Post-Translational↗

PubMed2023

Proteomics from compartment-specific APEX2 labeling in Mycobacterium tuberculosis reveals Type VII secretion substrates in the cell wall.

The cell wall of mycobacteria plays a key role in interactions with the environment. Its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites, from ions to lipids to proteins. Identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that although chemical labeling of live cells did not exclusively label surface proteins, protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis accurately identified the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.

Mycobacterium tuberculosis↗

PubMed1989

Inhibitors of asparagine-linked oligosaccharide processing alter the kinetics of the nicotinic acetylcholine receptor.

We used selective inhibitors of the asparagine-linked oligosaccharide processing pathway to study the effect of sugar trimming on the functional properties of the nicotinic acetylcholine (ACh) receptor expressed in clonal mammalian BC3H-1 cells. Inhibitors of initial steps of the processing pathway (1-deoxynojirimycin[DNJ] and castanospermine[CS]) reduced the density of ACh receptors on the cell surface (3- to 5-fold) but their responsiveness to ACh was more reduced (5- to 10-fold). These results suggest that the function of the ACh receptor was altered. When the ACh receptors were expressed in the presence of DNJ or CS, analysis of ACh-evoked single-channel currents (-100 mV and 11 degrees C) revealed an approximate threefold reduction in the opening rate (control: 600-650 s(-1)), treated: 130-250 s(-1)) and an approximate twofold reduction in the rate of agonist dissociation (control: 900-1,000 s(-1), treated: 400-500 s(-1)). In addition, the proportion of brief duration bursts (tau = 50-100 microseconds) was increased (1.5- to 2-fold) by treatments with DNJ or CS. In contrast, an inhibitor of a late processing step (swainsonine) did not produce such alterations. The single-channel conductance was not altered by any of the three inhibitors, and the slopes of log-log dose-response curves at low concentrations and desensitization did not appear to be affected. Each inhibitor altered the electrophoretic mobility of the ACh receptor subunits. We conclude that early sugar trimming can influence the kinetics of the nicotinic ACh receptor in BC3H-1 cells.

1-Deoxynojirimycin↗

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↗