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PubMed2024

Lineage-specific splicing regulation of MAPT gene in the primate brain.

Divergence of precursor messenger RNA (pre-mRNA) alternative splicing (AS) is widespread in mammals, including primates, but the underlying mechanisms and functional impact are poorly understood. Here, we modeled cassette exon inclusion in primate brains as a quantitative trait and identified 1,170 (∼3%) exons with lineage-specific splicing shifts under stabilizing selection. Among them, microtubule-associated protein tau (MAPT) exons 2 and 10 underwent anticorrelated, two-step evolutionary shifts in the catarrhine and hominoid lineages, leading to their present inclusion levels in humans. The developmental-stage-specific divergence of exon 10 splicing, whose dysregulation can cause frontotemporal lobar degeneration (FTLD), is mediated by divergent distal intronic MBNL-binding sites. Competitive binding of these sites by CRISPR-dCas13d/gRNAs effectively reduces exon 10 inclusion, potentially providing a therapeutically compatible approach to modulate tau isoform expression. Our data suggest adaptation of MAPT function and, more generally, a role for AS in the evolutionary expansion of the primate brain.

tau Proteins↗

PubMed1993

Site-specific initiation of transcription by RNA polymerase II.

RNA polymerase II initiates transcription at specific DNA sequences. Studies using sequence analysis and molecular genetics suggest a simple and universal model of start-site selection by RNA polymerase II. Two consensus sequences occur at fixed positions in promoters from higher eukaryotes and their viruses: the TATA box around -30 and the initiator at the start site of transcription. Both consensus sequences function as positioning elements that control site-specific initiation. As a first step during initiation, the basal transcription factor TFIID binds to the TATA box; regulatory transcription factors can tether TFIID bind to the TATA box; regulatory transcription factors can tether TFIID to promoters without a consensus TATA box. TFIID then directs the assembly of other basal transcription factors and RNA polymerase II into a preinitiation complex. Finally, RNA polymerase II searches for the best match to the initiator consensus about 30 base pairs downstream of the TATA box to select the exact start site. The transcriptional activity of a start-site sequence generally correlates with its similarity to the initiator consensus, suggesting that there is only one type of initiator.

Base Sequence↗

PubMed1991

Transfer of yeast artificial chromosomes from yeast to mammalian cells.

Human DNA can be cloned as yeast artificial chromosomes (YACs), each of which contains several hundred kilobases of human DNA. This DNA can be manipulated in the yeast host using homologous recombination and yeast selectable markers. In relatively few steps it is possible to make virtually any change in the cloned human DNA from single base pair changes to deletions and insertions. In order to study the function of the cloned DNA and the effects of the changes made in the yeast, the human DNA must be transferred back into mammalian cells. Recent experiments indicate that large genes can be transferred from the yeast host to mammalian cells in tissue culture and that the genes are transferred intact and are expressed. Using the same methods it may soon be possible to transfer YAC DNA into the mouse germ line so that the expression and function of genes cloned in YACs can be studied in developing and adult mammalian animals.

Animals↗

PubMed1980

Evaluation of functional capacity in activities of daily living in 70-year-old men and women.

Various activities of daily living of importance for an independent and active life were studied in 70-year-old systematically selected men (n = 190) and women (n = 229) recruited from a population study. The tests concluded basal movements in the upper extremities in hygiene and dressing activities, ability to rise from a stool, function in the kitchen e.g. reaching shelves and tasks including pronation and supination of the forearm and measurements of comfortable walking speed. In a selected subsample, tests of ability to climb various step-heights and a manual ability test were also performed. As regards basal movements in the upper extremities in hygiene and dressing activities, the hand-to-toe test caused most problems. A minority of both sexes had difficulty rising from a stool and in performing the pronation-supination tests. Problems in the test of reach were mainly correlated to the height of the subject, presence of arthritic disorders, and positional vertigo. The mean walking speed was in both sexes lower than that recommended in Sweden as the norm for pedestrians at signalized intersections. All but one subject could climb up and down a 40 cm step using a handrail. Without using handrail one-fifth of the women but only a few men had problems at this step-height but all managed the 30 cm step. Both sexes showed a decline in manual dexterity compared to 20-30-year-old subjects. Correlations were found between muscle strength, step test results and walking speed as well as between walking speed and leisure-time physical activity. There was also a correlation between problems in handling the plug in the manual ability test and strength in the key-grip.

Activities of Daily Living↗

PubMed1995

Progesterone receptor structure and function altered by geldanamycin, an hsp90-binding agent.

The assembly of progesterone receptor (PR) heterocomplexes in vitro involves at least eight components of the molecular chaperone machinery, and as earlier reports have shown, these proteins exhibit complex, dynamic, but ordered, interactions with one another and PR. Using the selective hsp90 binding agent geldanamycin (GA), we have found that PR assembly in vitro can be arrested at a previously observed intermediate assembly step. Like mature PR complexes, the intermediate complexes contain hsp90, but they differ from mature complexes by the presence of hsp70, p60, and p48 and the absence of immunophilins and p23. Arrest of PR assembly is likely due to GA's ability to directly block binding of p23 to hsp90. An important functional consequence of GA-mediated assembly arrest in vitro is the inability of the resulting PR complexes to bind progesterone, despite the presence of hsp90 in the receptor complexes. The biological significance of the in vitro observations is demonstrated by GA's ability to (i) rapidly block PR's hormone binding capacity in intact cells and (ii) alter the composition of COS cell PR complexes in a manner similar to that observed during in vitro reconstitutions. An updated model for the cyclic assembly pathway of PR complexes that incorporates the present findings with earlier results is presented.

Animals↗

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↗

PubMed1994

Dissociation of selective renal venous plasma endothelin-1 activity and corresponding plasma renin activity in a patient with hypertension, severe stenosis of unilateral renal artery and ipsilaterally decreased renal function.

Plasma endothelin-1 (ET-1)-like immunoreactivity in selective renal venous blood and peripheral venous blood was measured and compared with plasma renin activity and aldosterone level in a 26-year-old woman with suspected renovascular hypertension due to stenosis of the left renal artery. Although there was no significant step-up in plasma renin activity between the right and the left renal vein, left/right ratio of plasma ET-1 in the renal vein was 2.18. This case suggests that ET-1 may be one of the hypertensive mechanisms of suspected renovascular hypertension in addition to the renin-angiotensin system.

Adult↗

PubMed1995

Hyperresponse in calcium-induced insulin release from electrically permeabilized pancreatic islets of diabetics GK rats and its defective augmentation by glucose.

In spontaneously diabetic GK rats, insulin secretion from pancreatic beta cells in response to glucose is selectively impaired, probably due to deficient intracellular metabolism of glucose and impaired closure of KATP channels during glucose stimulation. By using electrically permeabilized islets of GK rats, we explored the functional modulations in exocytotic steps distal to the rise in [Ca2+]i in the diabetic condition. At 30 nmol/l Ca2+ (basal conditions) insulin release was similar between GK and non-diabetic control Wistar rats. In response to 3.0 mumol/l Ca2+ (maximum stimulatory conditions), insulin release was significantly augmented in permeabilized GK islets (p < 0.01). Raising glucose concentrations from 2.8 to 16.7 mmol/l further augmented insulin release induced by 3.0 mumol/l Ca2+ from permeabilized control islets (p < 0.001), but had no effect on that from permeabilized GK islets. The stimulatory effect of glucose on insulin release from permeabilized control islets was partly inhibited by 2,4-dinitrophenol, an inhibitor of mitochondrial oxidative phosphorylation (p < 0.01). The hyperresponse to Ca2+ in GK islets may play a physiologically compensatory role on the putative functional impairment both in [Ca2+]i rise and energy state in response to glucose in diabetic beta cells, and may explain the relative preservation of insulin release induced by non-glucose depolarizing stimuli, such as arginine, from pancreatic islets in non-insulin-dependent diabetes mellitus.

2,4-Dinitrophenol↗

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↗

PubMed1995

Isolation and characterization of mutants impaired in the selective degradation of peroxisomes in the yeast Hansenula polymorpha.

We have isolated a collection of peroxisome degradation-deficient (Pdd-) mutants of the yeast Hansenula polymorpha which are impaired in the selective autophagy of alcohol oxidase-containing peroxisomes. Two genes, designated PDD1 and PDD2, have been identified by complementation and linkage analyses. In both mutant strains, the glucose-induced proteolytic turnover of peroxisomes is fully prevented. The pdd1 and pdd2 mutant phenotypes were caused by recessive monogenic mutations. Mutations mapped in the PDD1 gene appeared to affect the initial step of peroxisome degradation, namely, sequestration of the organelle to be degraded by membrane multilayers. Thus, Pdd1p may be involved in the initial signalling events which determine which peroxisome will be degraded. The product of the PDD2 gene appeared to be essential for mediating the second step in selective peroxisome degradation, namely, fusion and subsequent uptake of the sequestered organelles into the vacuole. pdd1 and pdd2 mutations showed genetic interactions which suggested that the corresponding gene products may physically or functionally interact with each other.

Autophagy↗

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↗

PubMed1995

The Fusobacterium nucleatum major outer-membrane protein (FomA) forms trimeric, water-filled channels in lipid bilayer membranes.

The pore-forming activity of the major outer-membrane protein FomA of the anaerobic Fusobacterium nucleatum was studied in artificial lipid bilayer membranes. FomA was isolated from F. nucleatum strains Fev1, ATCC 10953, and ATCC 25586 by extraction with lithium dodecyl sulfate and lithium chloride and had an apparent molecular mass of about 40 kDa. When solubilized at low temperatures, the protein ran with an apparent molecular mass of about 62 kDa on SDS/PAGE. Cross-linking experiments and two-dimensional SDS/PAGE gave evidence that the 62-kDa protein band represented the trimeric form of FomA. The protein trimers were susceptible to SDS and temperature. The stability of the porin trimers varied among the strains. The properties of the FomA channels were studied in reconstitution experiments with black lipid bilayer membranes. The F. nucleatum porins formed channels with single-channel conductances in the range 0.66-1.30 nS in M KCl. The single-channel conductance was a function of the mobilities of the ions present in the aqueous solution bathing the bilayer membrane. This means that FomA forms general diffusion channels since (a) the conductance showed a linear dependence on the salt concentration, (b) the ion selectivity was small and varied for the three strains, and (c) the channels did not exhibit any binding site for maltotriose or triglycine. The water-filled channel was voltage dependent, and conductance decrements were observed at transmembrane potentials of +/- 50 mV. The conductance decrement steps were about one-third of the total conductance of a functional unit in its fully 'open' state. This strongly suggests that the trimer is the functional unit of the porin.

Bacterial Outer Membrane Proteins↗

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↗

PubMedDate not supplied

Genetic probes of ribosomal RNA function.

We have used a genetic approach to uncover the functional roles of rRNA in protein synthesis. Mutations were constructed in a cloned rrn operon by site-directed mutagenesis or isolated by genetic selections following random mutagenesis. We have identified mutations that affect each step in the process of translation. The data are consistent with the results of biochemical and phylogenetic analyses but, in addition, have provided novel information on regions of rRNA not previously investigated.

Base Sequence↗

PubMed1993

[The use of monoclonal antibodies in the study of ion transporting pumps].

The active ion transport is related to some vital functions for the normal eukaryotic cell metabolism. The study of transport mechanism and its regulation is a fundamental problem in cellular biology. The production of monoclonal antibodies (AcM) let us to have a probe, with large specificity, for the study of the localization, distribution and function of carrier proteins in the epithelium, as well as the study of its molecular structure, synthesis and assembling in the cell. The quality of a monoclonal antibody depends of the proper selection of each step to follow in the course of its production. In this article, we examine the strategy more currently used in the production of monoclonal antibodies and its direct use in the ionics pump's morphological, biochemical and physiological study.

Animals↗