Analyzing the Interaction of RseA and RseB, the Two Negative Regulators of the ��E Envelope Stress Response, Using a Combined Bioinformatic and Experimental Strategy

The Escherichia coli envelope stress response is controlled by the alternative sigma factor, σ^E, and is induced when unfolded outer membrane proteins accumulate in the periplasm. The response is initiated by sequential cleavage of the membrane-spanning antisigma factor, RseA. RseB is an important negative regulator of envelope stress response that exerts its negative effects onσ^E activity through its binding to RseA. In this study, we analyze the interaction between RseA and RseB. We found that tight binding of RseB to RseA required intact RseB. Using programs that performed global and local sequence alignment of RseB and RseA, we found regions of high similarity and performed alanine substitution mutagenesis to test the hypothesis that these regions were functionally important. This protocol is based on the hypothesis that functionally dependent regions of two proteins co-evolve and therefore are likely to be sequentially conserved. This procedure allowed us to identify both an N-terminal and C-terminal region in RseB important for binding to RseA. We extensively analyzed the C-terminal region, which aligns with a region of RseA coincident with the major RseB binding determinant in RseA. Both allele-specific suppression analysis and cysteine-mediated disulfide bond formation indicated that this C-terminal region of similarity of RseA and RseB identifies a contact site between the two proteins. We suggest a similar protocol can be successfully applied to pairs of non-homologous but functionally linked proteins to find specific regions of the protein sequences that are important for establishing functional linkage.The Escherichia coli σ^E-mediated envelope stress response is the major pathway to ensure homeostasis in the envelope compartment of the cell (1-3). σ^E regulon members encode periplasmic chaperones and proteases, the machinery for inserting β-barrel proteins into the outer membrane and components controlling the synthesis and assembly of LPS (4-6). This pathway is highly conserved among γ-proteobacteria (6).The σ^E response is initiated when periplasmic protein folding and assembly is compromised (7-9). During steady state growth, σ^E is inhibited by its antisigma factor, RseA, a membrane-spanning protein whose cytoplasmic domain binds to σ^E with picomolar affinity (10-13). Accumulation of unassembled porin monomers serves as a signal to activate the DegS protease to cleave RseA in its periplasmic domain (14, 15). This initiates a proteolytic cascade in which RseP cleaves periplasmically truncated RseA near or within the cytoplasmic membrane to release the RseA_cytoplasmic-σ^E complex, and cytoplasmic ATP-dependent proteases complete the degradation of RseA thereby releasing active σ^E (16-19).RseB, a second negative regulator of the envelope stress response (11, 20, 21), binds to the periplasmic domain of RseA with nanomolar affinity. RseB is an important regulator of the response (2, 22, 23). It prevents RseP from degrading intact RseA, thereby ensuring that proteolysis is initiated only when the DegS protease is activated by a stress signal (21). Additionally, RseB prevents activated DegS from cleaving RseA, suggesting that interaction of RseB with RseA must be altered before the signal transduction cascade is activated (23).The goal of the present studies was to explore how RseB binds to RseA. The interaction partner of RseB is the unstructured periplasmic domain of RseA (RseA-peri). Within RseA-peri, amino acids ∼169-186 constitute a major binding determinant to RseB (23, 24). This peptide alone binds RseB with 6 μm affinity, and deleting this region abrogates binding to RseB (23). Additional regions of RseA-peri also contribute to RseB binding, as intact RseA-peri binds with 20 nm affinity to RseB (23). Much less is known about the regions of RseB required for interaction with RseA. RseB is homodimeric two-domain protein, whose large N-terminal domain shares structural homology with LolA, a protein that transports lipoproteins to outer membrane (24, 25). The smaller C-terminal domain is connected to the N-terminal domain by a linker, and the two domains share a large interface, which may facilitate interdomain signaling. Glutaraldehyde cross-linking studies indicate that the C-terminal domain interacts with RseA, but the regions of interaction were not identified (25).In the present report, we study the interaction of RseB and RseA. We establish that both domains of RseB interact with RseA-peri. Using a global sequence alignment, we discovered several regions in RseA and RseB that had high sequence similarity, despite the low overall sequence similarity between these two proteins, a finding that was independently confirmed by a local sequence similarity algorithm. This suggested that these regions were functionally dependent, and we performed a set of mutagenesis experiments designed to test this idea. Our studies of the binding properties of these mutants revealed that regions in both the N terminus and C terminus of RseB modulate interaction with RseA. Moreover, genetic suppression analysis and cysteine-mediated disulfide bond formation suggest that the region of RseA/B with highest similarity (RseA residues 165-191 (major binding determinant in RseA) and RseB residues 233-258) are interacting partners.     

Review of the fauna of Rotatoria,Cladocera, and Copepoda of the basin of the Anadyr’ River

The zooplankton composition is studied in the thermokarst, glacial and meteorite lakes, channels, former riverbeds, and hollows in the basin of Anadyr’. We found 174 taxa: 78, Rotatoria, 55, Cladocera, and 41, Copepoda. The most diverse is the lake fauna: 51 taxa of Rotatoria, 48, Cladocera, and 37, Copepoda. The thermokarst Lake Maiorskoe hosts 68 taxa: 31, Rotatoria, 14, Cladocera, and 23, Copepoda, wheras the cold ultraoligotrophic Lake El’gygytgyn features only one species of Cyclop of the group scutifer Cyclops neymanae Strel., and Rotatoria and Cladocera are present as allochtonous forms. The Copepoda illustrate the relations of the Anadyr’ fauna with those of Europe, North America, and Japan.     

A double blind,randomized, placebo controlled study of the efficacy and safety of 5-Loxin<Superscript>®</Superscript>for treatment of osteoarthritis of the knee

Introduction

5-Loxin^® is a novel Boswellia serrata extract enriched with 30% 3-O-acetyl-11-keto-beta-boswellic acid (AKBA), which exhibits potential anti-inflammatory properties by inhibiting the 5-lipoxygenase enzyme. A 90-day, double-blind, randomized, placebo-controlled study was conducted to evaluate the efficacy and safety of 5-Loxin^® in the treatment of osteoarthritis (OA) of the knee.

Methods

Seventy-five OA patients were included in the study. The patients received either 100 mg (n = 25) or 250 mg (n = 25) of 5-Loxin^® daily or a placebo (n = 25) for 90 days. Each patient was evaluated for pain and physical functions by using the standard tools (visual analog scale, Lequesne''s Functional Index, and Western Ontario and McMaster Universities Osteoarthritis Index) at the baseline (day 0), and at days 7, 30, 60 and 90. Additionally, the cartilage degrading enzyme matrix metalloproteinase-3 was also evaluated in synovial fluid from OA patients. Measurement of a battery of biochemical parameters in serum and haematological parameters, and urine analysis were performed to evaluate the safety of 5-Loxin^® in OA patients.

Results

Seventy patients completed the study. At the end of the study, both doses of 5-Loxin^® conferred clinically and statistically significant improvements in pain scores and physical function scores in OA patients. Interestingly, significant improvements in pain score and functional ability were recorded in the treatment group supplemented with 250 mg 5-Loxin^® as early as 7 days after the start of treatment. Corroborating the improvements in pain scores in treatment groups, we also noted significant reduction in synovial fluid matrix metalloproteinase-3. In comparison with placebo, the safety parameters were almost unchanged in the treatment groups.

Conclusion

5-Loxin^® reduces pain and improves physical functioning significantly in OA patients; and it is safe for human consumption. 5-Loxin^® may exert its beneficial effects by controlling inflammatory responses through reducing proinflammatory modulators, and it may improve joint health by reducing the enzymatic degradation of cartilage in OA patients.

Trail Registration

(Clinical trial registration number: ISRCTN05212803.)     

α<Subscript>1</Subscript>-Thymosin, α<Subscript>2</Subscript>-interferon,and the LKEKK syntetic peptide inhibit the binding of the B subunit of the cholera toxin to intestinal epithelial cell membranes

The ¹²⁵I-labeled B-subunit of the cholera toxin ([125I]CT-B, specific activity of 98 Ci/mmol) was prepared. This subunit was shown to be bound to the membranes which were isolated from epithelial cells of a mucous tunic of the rat thin intestine with high affinity (K _d = 3.7 nM). The binding of the labeled protein was inhibited by the unlabeled α₂-interferon (IFN-α₂), α₁-thymosin, (TM-α₁), and the LKEKK synthetic peptide corresponding to the 16–20 sequence of TM-α₁ and the 131–135 sequence of human IFN-α₂ (Ki 1.0, 1.5, and 2.0 nM, respectively), whereas the KKEKL unlabeled synthetic peptide did not inhibit the binding (K _i > 100 μМ). The LKEKK peptide and CT-B were shown to dose-dependently increase an activity of the soluble guanylate cyclase (sGC) in the concentration range from 10 to 1000 nM. Thus, the binding of TM- α₁, IFN-α₂, and the LKEKK peptide to the CT-B receptor on a surface of the epithelial cells of the mucous tunic of the rat thin intestine resulted in an increase in the intracellular level of cGMP.     

Plastome phylogenomics of Allaeanthus,Broussonetia and Malaisia (Dorstenieae,Moraceae) and the origin of B. × kazinoki

Species of Broussonetia have been essential in the development of papermaking technology. In Japan and Korea, a hybrid between B. monoica and B. papyrifera (= B. × kazinoki) known as kōzo and daknamu is still the major source of raw materials for making traditional paper washi and hanji, respectively. Despite their cultural and practical significance, however, the origin and taxonomy of kōzo and daknamu remain controversial. Additionally, the long-held generic concept of Broussonetia s.l., which included Sect. Allaeanthus and Sect. Broussonetia, was challenged as phylogenetic analyses showed Malaisia is sister to the latter section. To re-examine the taxonomic proposition that recognizes Allaeanthus, Broussonetia, and Malaisia (i.e., Broussonetia alliance), plastome and nuclear ribosomal DNA (nrDNA) sequences of six species of the alliance were assembled. Characterized by the canonical quadripartite structure, genome alignments and contents of the six plastomes (160,121–162,594 bp) are highly conserved, except for the pseudogenization and/or loss of the rpl22 gene. Relationships of the Broussonetia alliance are identical between plastome and nrDNA trees, supporting the maintenance of Malaisia and the resurrection of Allaeanthus. The phylogenomic relationships also indicate that the monoecy in B. monoica is a derived state, possibly resulting from hybridization between the dioecious B. kaempferi (♀) and B. papyrifera (♂). Based on the hypervariable ndhF-rpl32 intergenic spacer selected by sliding window analysis, phylogeographic analysis indicates that B. monoica is the sole maternal parent of B. × kazinoki and that daknamu carries multiple haplotypes, while only one haplotype was detected in kōzo. Because hybridizations between B. monoica and B. papyrifera are unidirectional and have occurred rarely in nature, our data suggest that daknamu might have originated via deliberate hybrid breeding selected for making hanji in Korea. On the contrary, kōzo appears to have a single origin and the possibility of a Korean origin cannot be ruled out.

     

Rarity,Species Richness,and the Threat of Extinction—Are Plants the Same as Animals?

Assessment of conservation status is done both for areas or habitats and for species (or taxa). IUCN Red List categories have been the principal method of categorising species in terms of extinction risk, and have been shown to be robust and helpful in the groups for which they have been developed. A recent study highlights properties associated with extinction risk in flowering plants, focusing on the species-rich hot spot of the Cape region of South Africa, and concludes that merely following methods derived from studies of vertebrates may not provide the best estimates of extinction risk for plants. Biology, geography, and history all are important factors in risk, and the study poses many questions about how we categorise and assess species for conservation priorities.     

Flexibility in the order of action and in the enzymology of the nuclease, polymerases, and ligase of vertebrate non-homologous DNA end joining： relevance to cancer, aging, and the immune system

Nonhomologous DNA end joining （NHEJ） is the primary pathway for repair of double-strand DNA breaks in human cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the site of damage, resulting in the loss of information there. NHEJ does not restore the lost information and may resect additional nucleotides during the repair process. The ability to repair a wide range of overhang and damage configurations reflects the flexibility of the nuclease, polymerases, and ligase of NHEJ. The flexibility of the individual components also explains the large number of ways in which NHEJ can repair any given pair of DNA ends. The loss of information locally at sites of NHEJ repair may contribute to cancer and aging, but the action by NHEJ ensures that entire segments of chromosomes are not lost.     

Chemotaxis of the unicellular green algaDunaliella salina and the ciliatedTetrahymena pyriformis—effects of glycine,lysine, and alanine,and their oligopeptides

Chemotactic properties of amino acids (L-alanine, glycine and L-lysine) and their oligopeptides (10^–6M) and binding sites to these ligands were investigated in two unicellular models, the heterotrophicTetrahymena pyriformis and the auxotrophicDunaliella salina. Chemotaxis ofDunaliella induced by simple amino acids and their derivatives demonstrated that binding sites (receptors) for food molecules are not only present in the membrane but are also able to induce their basic physiological response. InTetrahymena, substances with special molecular structure and properties (polar, hydrophilic character of the signal peptide chain)-5-L-Lys, 5-Glywere required for chemoattraction, other peptides tested, lacking the required structure, were repellent. Divergences in chemotaxis and binding assays of both species suggest that trends of functional and binding parameters do not run parallel at this level of evolution.     

Description of a new Brazilian Paraportanus and key to the species of the genus (Insecta,Hemiptera,?Cicadellidae,Portanini)

Paraportanus longispinus,, a new leafhopper species from Roraima and Amazonas States, North Brazil, is described and illustrated. The new species can be recognized by the male genital features, especially the distal third of ventral margin of the pygofer with a dentiform short process; plates distinctly longer than pygofer, extending posteriorly beyond pygofer by approximately 1/3 of their length and aedeagus with one pair of spiniform process long crossed and directed ventrally. A checklist and key to males of all known Paraportanus species is provided.     

Cloning,characterisation and expression of the α-tubulin genes of the leech,Hirudo medicinalis

We have isolated two α-tubulin cDNAs from the leech, Hirudo medicinalis. Both encode putative proteins of 451 amino-acids which differ from each other at only two positions. Southern blotting suggests that there are only two α-tubulin genes in the leech. The genes contain two introns and, because of the extremely high homology of the nucleotide sequence from the second intron to the end of the genes, we have inferred that a gene conversion event about 9.5 million years ago has homogenised the Hirudo α-tubulin sequences. Using in situ hybridisation to tissue sections, we have shown that the two genes are probably expressed in all neurons of the leech ganglia and that their spatial distribution remains unchanged during neuronal regeneration. The deduced amino-acid sequences of the leech α-tubulins show that they have greatest similarity to those from a platyhelminth, echiuran and mollusc with rather less to arthropod α-tubulins. The protein sequences of the leech α-tubulins have been compared with representatives of those from across all phyla to determine if any specific feature labels certain isotypes of tubulin for neuronal expression.     

Distribution of α-N-acetylgalactosaminidases among marine bacteria of the phylum <Emphasis Type="Italic">Bacteroidetes</Emphasis>, epiphytes of marine algae of the Seas of Okhotsk and Japan

Occurrence of α-N-acetylgalactosaminidases among 177 strains of marine bacteria of the phylum Bacteroidetes, epiphytes of marine algae growing on the littoral of the Seas of Okhotsk and Japan, was studied. About 36% of the isolates studied contained α-N-acetylgalactosaminidase. All of the bacteria of the genus Arenibacter (species A. latericius, A. certesii, and A. palladensis), irrespective of the source of isolation, synthesized this enzyme. The greatest number of α-N-acetylgalactosaminidase producers was found among the isolates from the algae Neosiphonia japonica, Acrosiphonia sonderi, and Ulva fenestrata sampled in the Cove of Trinity, Posyet Bay, the Sea of Japan. These were mainly bacteria of the genera Zobellia (50%) and Maribacter (58%). Among the epibionts studied, the bacteria Arenibacter latericius KMM 3523, an epiphyte of the brown alga Chorda filum from the Sea of Okhotsk, and Cellulophaga sp. KMM 6488, an epiphyte of the green alga Acrosiphonia sonderi from the Sea of Japan, were marked as the most promising sources of the enzyme. The results of this study showed that aerobic nonpathogenic marine Bacteroidetes, algal associants not requiring special cultivation conditions, are the promising, economical, and ecologically pure sources of unique and biotechnologically significant α-N-acetylgalactosaminidases.     

Analysis of the amide <Superscript>15</Superscript>N chemical shift tensor of the C<Subscript>α</Subscript> tetrasubstituted constituent of membrane-active peptaibols,the α-aminoisobutyric acid residue,compared to those of di- and tri-substituted proteinogenic amino acid residues

In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis of ¹⁵N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols, i.e. polypeptides rich in α-aminoisobutyric acid (Aib), the ¹⁵N chemical shift tensor of this C_α-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues. Here we present an experimental investigation on the ¹⁵N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted amino acids. A ¹⁵N chemical shift-¹H-¹⁵N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main tensor elements for the structural analysis of this membrane peptide.     

Assay, purification, properties and mechanism of action of γ-glutamylcysteine synthetase from the liver of the rat and Xenopus laevis

1. An improved radioassay for glutathione synthetase and gamma-glutamylcysteine synthetase was developed. 2. Xenopus laevis liver gamma-glutamylcysteine synthetase was purified 324-fold by saline-bicarbonate extraction, protamine sulphate precipitation, CM-cellulose and DEAE-cellulose column chromatography, and gel filtration. 3. Rat liver gamma-glutamylcysteine synthetase was purified 11400-fold by a procedure similar to that employed for the Xenopus laevis enzyme. 4. Rat liver gamma-glutamylcysteine synthetase activity was inhibited by GSH and activated by glycine. These effects, which were not found in the enzyme from Xenopus laevis, may have a regulatory significance. 5. Isotope-exchange experiments revealed fundamental differences in the partial reactions catalysed by the rat and Xenopus laevis synthetases. The enzyme from Xenopus laevis appears to follow a Bi Bi Uni Uni Ping Pong mechanism, with glutamyl-enzyme as intermediate before the addition of cysteine and the release of gamma-glutamylcysteine. The results for the rat liver enzyme are consistent with a Tri Tri sequential mechanism.     

Anaerobic microbial dehalogenation of organohalides — state of the art and remediation strategies

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Cell-free expression,purification, and characterization of the functional β<Subscript>2</Subscript>-adrenergic receptor for multianalyte detection of β-agonists

Large-scale expression of β₂-adrenergic receptor (β₂-AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused β-adrenergic agonists (β-agonists). Cell-based heterologous expression systems have many critical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional β₂-AR in a cell-free system based on Escherichia coli extracts. A codon-optimized porcine β₂-AR gene (codon adaptation index: 0.96) suitable for high expression in E. coli was synthesized and transcribed to the cell-free system, which contributed to increase the expression up to 1.1 mg/ml. After purification using Ni-affinity chromatography, the bioactivity of the purified receptor was measured by novel enzyme-linked receptor assays. It was determined that the relative affinities of the purified β2-AR for β-agonists in descending order were as follows: clenbuterol > salbutamol > ractopamine. Moreover, their IC₅₀ values were 45.99, 60.38, and 78.02 μg/liter, respectively. Although activity of the cell-free system was slightly lower than activity of systems based on insect and mammalian cells, this system should allow production of β2-AR in bulk amounts sufficient for the development of multianalyte screening methods for detecting β-agonist residues.     

To speciate,or not to speciate? Resource heterogeneity,the subjectivity of similarity,and the macroevolutionary consequences of niche‐width shifts in plant‐feeding insects

Coevolutionary studies on plants and plant‐feeding insects have significantly improved our understanding of the role of niche shifts in the generation of new species. Evolving plant lineages essentially constitute moving islands and archipelagoes in resource space, and host shifts by insects are usually preceded by colonizations of novel resources. Critical to hypotheses concerning ecological speciation is what happens immediately before and after colonization attempts: if an available plant is too similar to the current host(s), it simply will be incorporated into the existing diet, but if it is too different, it will not be colonized in the first place. It thus seems that the probability of speciation is maximized when alternative hosts are at an ‘intermediate’ distance in resource space. In this review, I wish to highlight the possibility that resource similarity and, thus, the definition of ‘intermediate’, are subjective concepts that depend on the herbivore lineage's tolerance to dietary variation. This subjectivity of similarity means that changes in tolerance can either decrease or increase speciation probabilities depending on the distribution of plants in resource space: insect lineages with narrow tolerances are likely to speciate by ‘island‐hopping’ on young, species‐rich plant groups, whereas more generalized lineages could speciate by shifting among resource archipelagoes formed by higher plant taxa. Repeated and convergent origins of traits known to broaden or to restrict host‐plant use in multiple different insect groups provide opportunities for studying how tolerance and resource heterogeneity may interact to determine speciation rates.     

Chitinozoan zonation of the Late Ordovician and the Early Silurian of the island of Anticosti, Québec, Canada

A reappraisal of chitinozoan distribution across the Ordovician-Silurian boundary on the Island of Anticosti has led to the recognition of a new zone, the Ancyrochitina ellisbayensis biozone, in the uppermost part of the Ellis Bay Formation. This biozone lies between the well defined Upper Ordovician Spinachitina taugourdeaui biozone and the lowest Silurian (Rhuddanian) Plectochitina nodifera biozone of the Becscie Formation. The occurrence of such diagnostic species as P. nodifera, Belonechitina postrobusta, Conochitina electa and Ancyrochitina ramosaspina in the Lower Silurian of Anticosti points to a close similarity to faunas in Estonia and north Latvia and indicates an age ranging from the Parakidograptus acuminatus to the Coronograptus cyphus in terms of graptolite zones. The chitinozoan biozonation harmonizes with that based on conodonts and, to a lesser extent, with the known graptolite faunal succession. Five new species: Ancyrochitina ellisbayensis sp. nov., Clathrochitina postconcinna sp. nov., Conochitina gunriveris sp. nov., Clathrochitina perexilis sp. nov., Bursachitina basiconcava sp. nov. and three species in open nomenclature are described.     

A DFT study of the catalytic pyrolysis of benzaldehyde on ZnO, γ-Al<Subscript>2</Subscript>O<Subscript>3</Subscript>, and CaO models

The catalytic pyrolysis pathways of carbonyl compounds in coal were systematically studied using density functional theory (DFT), with benzaldehyde (C₆H₅CHO) employed as a coal-based model compound and ZnO, γ-Al₂O₃, and CaO as catalysts. The results show that the products of both pyrolysis and catalytic pyrolysis are C₆H₆ and CO. However, the presence of any of the catalysts changes the reaction pathway and reduces the energy barrier, indicating that these catalysts promote C₆H₅CHO decomposition.

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Graphical abstract The presence of catalysts changes the reaction pathway and the energy barrier decreases in the order Ea (no catalyst)> Ea (CaO)> Ea (γ-Al₂O₃)> Ea (ZnO), indicating that these catalysts promote C₆H₅CHO decomposition.

     

Comparative Study of the Absorption,Plasma Levels,and Urinary Excretion of the “New” and the “Old” Lanoxin

A comparative study was performed of the absorption, the plasma level at equilibrium, and the urinary excretion of digoxin using two types of Lanoxin tablets, those produced before and after the 1972 alteration of the tablet manufacture.After a single dose the absorption rate of the new tablets was about twice as great as the old, both in young subjects and in the elderly patients. There were no significant differences in the plasma levels of digoxin for the two tablets 15 hours after the last administration in patients on an equal maintenance dose. The urinary excretion of digoxin increased about 40% when the “old” Lanoxin was replaced by the “new.” In elderly patients a daily dose of 0·125 mg twice daily of the new tablets should be sufficient to reach the therapeutic range. Young people need a higher dosage. If the kidney function is reduced by as much as 50% the dose should be reduced.