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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↗
PubMed1980

Globin mRNA in Friend cells: its structure, function and synthesis.

Accumulation of haemoglobin in induced MEL cells begins with the activation of transcription of the globin genes. Though much has been learned of cellular events affecting expression of the globin genes, e.g., from noninducible variants of MEL cells and cell fusion between MEL cells and other cell types, there is at present no in vitro system available that would permit more detailed study of the molecular events leading to transcription of the globin genes. Presumably, with the availability of cloned chromosomal genes such systems will soon be found. The products of transcription detected in induced MEL cells are 15 S and 11 S species which are precursor forms of beta- and alpha-globin mRNA, respectively. An unmodified primary transcript has not been detected. The 15 S species possesses a fully methylated 'cap' 1 structure the 5' end and poly(A) at the 3' end. Conceivably, there could be cleaving or splicing events preceding the 'capping' and polyadenylation, but all these reactions must occur extremely rapidly, since with a t 1/2 approx. 2 min a large proportion of the 15 S beta-globin RNA must be newly synthesised. It also contains the two intervening sequences found in the chromosomal genes. Selective processing occurs since from pulse and pulse-chase experiments most if not all of the 15 S beta-globin RNA is processed to mature 10 S beta-globin RNA very rapidly, whereas less than 10% of newly synthesised nuclear RNA (HnRNA) leaves the nucleus, the remainder being hydrolysed in the nucleus with a t 1/2 approx. 20 min. Perhaps rapid processing permits efficient transport to the cytoplasm. Further processing occurs in steps; apparently the large intervening sequence is removed first followed by the small intervening sequence. These steps do not appear to be rate limiting events and these sequences have not been detected separately from the 15 S beta-globin RNA. Such results and the wide divergency of intervening sequences, suggest that the intervening sequences per se play no essential function in the cell, though their presence in the nuclear transcript appears to necessary for processing to the mRNA. The selective processing accounts for more increase of the globin RNA in MEL cells, and further accumulation occurs by virtue of the stability of globin mRNA (t 1/2 approx. 17 h) compared with the bulk of poly(A)-RNA (t 1/2 approx. 3 h). It would appear, however, that specific destabilization of a class of stable mRNA (t 1/2 approx. 35 h) is necessary to allow globin mRNA to asccount for 90% of the mRNA population in reticulocytes.

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↗
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↗
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↗