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PubMed1978

Ascorbic acid and L-gulonolactone oxidase in lagomorphs.

1. The activity of L-gulonolactone oxidase (EC 1.1.3.8) in the liver of eastern cottontail rabbits (Sylvilagus floridanus) is about 10-fold greater in winter than in summer. 2. L-gulonolactone oxidase activity is low and tissue ascorbate high during all seasons in snowshoe hares (Lepus americanus). 3. Liver contents of ascorbate fall to low levels in L. americanus fed on rabbit chow in the laboratory. 4. The activity of L-gulonolactone oxidase in liver of Sylvilagus and Oryctolagus is depressed by feeding high levels of L-ascorbic acid. 5. The New Zealand White breed of domestic rabbit (Oryctolagus cuniculus) has considerably higher levels of L-gulonolactone oxidase and liver ascorbate than does the Dutch breed. 6. In a wild population of Oryctolagus sampled in Australia L-gulonolactone oxidase levels were intermediate between those of the two domestic breeds and more variable than either.

Animals↗
PubMed2006

Efficient screening method of the thiopurine methyltransferase polymorphisms for patients considering taking thiopurine drugs in a Chinese Han population in Henan Province (central China).

BACKGROUND: Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzed the S-methylation of thiopurine drugs. TPMT activity exhibits an interindividual variability, mainly as a result of genetic polymorphism. Patients with intermediate or deficient TPMT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. We determined a cut-off concentration of the TPMT activity assay less than which genotyping of the TPMT gene should be performed. In addition, the influence of hemodialysis on TPMT activity in uremic patients was examined. METHODS: In 248 healthy subjects and 30 uremic patients, PCR-based methods were used to analyze the most common functional mutations TPMT2, 3A, 3B and 3C. A HPLC assay was used to measure erythrocyte TPMT activity in the whole population. RESULTS: Seven TPMT3C heterozygotes were identified, while TPMT2, 3A and 3B alleles were not detected in 248 healthy subjects. The frequency of TPMT3C allele was 1.4% (7/496). The TPMT activity in healthy subjects was normally distributed, ranged from 6.09 to 28.65 nmol/h/ml pRBC with a mean of 16.03 +/- 4.16 nmol/h/ml pRBC. The cut-off for high TPMT activity and intermediate TPMT activity was 10.07 nmol/h/ml pRBC. There were 19 intermediate activity healthy subjects (7.7%) and 229 high activity healthy subjects (92.3%), and no TPMT deficiency subject was found. All of the 229 healthy subjects with high activity had no mutant alleles, while 7 of the 19 subjects with intermediate activity had a mutant allele. Phenotypes were in good agreement with genotypes for 95% of subjects. The uremic patients were all homozygous for the wild-type allele whose TPMT activity was activated significantly before hemodialysis compared with TPMT activity after hemodialysis. CONCLUSIONS: We defined the cut-off values for the TPMT phenotyping assay at 10.07 nmol/h/ml pRBC, less than which additional genotyping elucidates the individual risk for drug therapy. In uremic patients, TPMT activity is increased by some uremic factors, and dialysis shifted their TPMT activity close to that of a healthy control group.

Adult↗
PubMed2002

Evaluation of 6-year application of the enzymatic colorimetric phenylalanine assay in the setting of neonatal screening for phenylketonuria.

BACKGROUND: Most reports on phenylketonuria (PKU) screening focused solely on the result of the initial investigation of the neonatal screening sample. The aim of this study was to evaluate an enzymatic phenylalanine (Phe) determination in the whole context spanning from the initial investigation over the recall period, up to the confirmation or exclusion of the disease. METHODS: Phe of dried blood spot specimens was analysed colorimetrically in a microtitre-plate assay based on the L-phenylalanine dehydrogenase reaction coupled with an intermediate electron acceptor system. This assay was evaluated for analytical variables and for neonatal PKU screening in a total number of 423,773 neonates during a 6-year period. RESULTS: Method validation with respect to linearity, precision (within-run CVs 3.4-4.2%, between-run CVs 6.2-10.4%), and accuracy fulfilled all requirements for a screening method. Mean Phe (+/-SD) of 130,000 healthy neonates was 84 (+/-22) micromol/l with a cut-off point (mean+3 SD) of 150 micromol/l. From 423,773 neonates, hyperphenylalaninemia was confirmed in 155 cases and further differentiated into PKU (41 cases, 27%), BH(4) deficiency (3, 2%), non-PKU HPA (67, 43%), transient neonatal HPA (28, 18%), and secondary HPA (16, 10%). The number of false-positives (recall-rate) was 0.23%, and no false-negatives were noted. CONCLUSIONS: Detailed studies over a period of 6 years including more than 400,000 neonates clearly show that the enzymatic assay is a reliable and sensitive method for neonatal screening of PKU. The proven prevalence of non-PKU HPA in the German population disclosed by the assay was twice as high as compared to the "Guthrie test" used previously. The growing use and application of tandem mass spectrometry in neonatal screening will not derogate the usefulness of the enzymatic assay in PKU screening in the foreseeable future. Careful analysis of our screening results and monitoring of all pathological samples resulted in an evidence-based flow chart for a rational PKU screening.

Amino Acid Metabolism, Inborn Errors↗
PubMed2003

Operation and control of a water supply system.

The control of water supply systems is becoming more important, since there are increasing requirements to improve operation. A need exists to model and simulate water supply systems so that their behavior can be fully understood and the total process optimized. This paper describes the simulation and control of a water supply system consisting of a sequence of pumping stations that deliver water through pipelines to intermediate storage reservoirs. The system is represented by dominant system variables that represent active and passive dynamical elements. The hydraulic models include the nonlinear coupling between flow rates and reservoir heads. The bisection numerical solution approach is used to obtain a roughness dependent friction coefficient. The whole system is simulated and the results are presented and compared with the real-time measured data. A water level controller using the robust polynomial H(infinity) optimization method by manipulating pump speed is obtained. The stochastic nature of the disturbance and loads is considered for controller design. The parametrized dynamic weighting functions of the design theory are selected to achieve the required control functions and robustness.

Journal Article↗
PubMed2002

A review of dicrocoeliosis of ruminants including recent advances in the diagnosis and treatment.

Despite its widespread presence among grazing ruminants, dicrocoeliosis, also known as "small liver fluke" disease, is poorly known and often underestimated by researchers and practitioners in many countries. This is primarily due to the multiple parasitic infections which affect ruminant livestock and mask the pathology of dicrocoeliosis, to the difficulties in diagnosing it with coprological techniques and, finally, to the few effective drugs found. Furthermore, the biological cycle of Dicrocoelium, which requires a snail and an ant as intermediate hosts, and the high number of ecological and epidemiological variables affecting the disease make it difficult to set up experimental designs to study dicrocoeliosis. In the past 50 years, many aspects of this disease have been broadly investigated (aetiology, life cycle, diffusion, epidemiology, pathogenesis and immunology) but its diagnosis and treatment still remain moot issues. Dicrocoeliosis often remains clinically undetected and its diagnosis is mostly based on adult dicrocoelia recovered in the liver post mortem or on egg detected at coprological examination. The prophylaxis of the small liver fluke has been difficult and unsatisfactory to date due to the complexity of its biological life cycle and epidemiology. Many anti-helminthic drugs are practically ineffective against dicrocoeliosis if used at the dosage recommended against other gastrointestinal helminths and lungworms. The most important aspects of the aetiology, biological cycle, spread, epidemiology and pathogenesis of dicrocoeliosis are reviewed and the recent advances in the diagnosis and treatment are focused on.

Animals↗
PubMed1996

[Hypoxic-ischemic encephalopathy of the full term newborn. Contribution of the electroencephalogram and trans-fontanelle echography to the prognostic evaluation. Report of 29 cases].

This study was aimed at assessing by EEG recording and cranial imaging the cerebral function of 29 full term newborns presenting with hypoxic-ischemic encephalopathy and at establishing a correlation between the results and the neurological outcome. A correlation between the Sarnar's classification and the neurological outcome was observed, except for the intermediate grade. In this case, impairment of the EEG was variable and neurological prognosis was sometimes evidenced by cranial imaging. Unfavorable neurological outcome occurred when thalamic lesions were present, independently of clinical signs and EEG abnormalities.

Brain Ischemia↗
PubMed2006

NMR characterization of 13C-benzene sorbed to natural and prepared charcoals.

We investigated how the NMR properties of uniformly 13C-labeled benzene molecules are influenced by sorption to charcoals produced in the laboratory and collected from the field following wildfires. Uniformly 13C-labeled benzene was sorbed to two charcoals produced in the laboratory at 450 and 850 degrees C. The chemical shift of benzene sorbed to the higher-temperature charcoal was 5-6 ppm lower than that of benzene sorbed to the lower-temperature charcoal. This difference was attributed to stronger diamagnetic ring currents (which cause a shift to lower ppm values) in the more condensed or "graphitic" high-temperature charcoal. The chemical shift of benzene sorbed to two charcoals collected from the field following wildfires indicated a degree of charcoal graphitization intermediate between that of the two laboratory-prepared charcoals. Variable contact time and dipolar dephasing experiments showed that the molecular mobility of sorbed benzene molecules increased with increasing charcoal graphitization, and also increased with increasing benzene concentration. We propose that the chemical shift displacement of molecules sorbed to charcoal could be used to identify molecules sorbed to black carbon in heterogeneous matrixes such as soils and sediments, and to establish how condensed or "graphitic" the black carbon is.

Adsorption↗
PubMed1998

New chemical descriptors relevant for the design of biologically active peptides. A multivariate characterization of 87 amino acids.

In this study 87 amino acids (AA.s) have been characterized by 26 physicochemical descriptor variables. These descriptor variables include experimentally determined retention values in seven thin-layer chromatography (TLC) systems, three nuclear magnetic resonance (NMR) shift variables, and 16 calculated variables, namely six semiempirical molecular orbital indices, total, polar, and nonpolar surface area, van der Waals volume of the side chain, log P, molecular weight, and four indicator variables describing hydrogen bond donor and acceptor properties, and side chain charge. In the present study, the data from a previous characterization of 55 AA.s from our laboratory have been extended with data for 32 additional AA.s and 14 new descriptor variables. The new 32 AA.s were selected to represent both intermediate and more extreme physicochemical properties, compared to the 20 coded AA.s. The new extended and updated principal property scales, the z-scales, were calculated and aligned to previously reported z(old)-scales. The appropriateness of the extended z-scales were validated by the use in quantitative sequence-activity modeling (QSAM) of 89 elastase substrate analogues and in a QSAM of 29 neurotensin analogues.

Amino Acids↗
PubMed1997

Intermediates produced in the reaction of chromium(VI) with dehydroascorbate cause single-strand breaks in plasmid DNA.

Ascorbate (vitamin C) is a biological reductant of the human carcinogen chromium(VI). The product of this reaction is presumed to be dehydroascorbate. However, we have found that chromium(VI) can also react with dehydroascorbate. This reaction was monitored by UV/ visible and electron paramagnetic resonance (EPR) spectroscopies. In sodium acetate buffer at pH 3.8, the reaction of chromium(VI) and excess dehydroascorbate produced chromium(V) and chromium(IV) intermediates. At high reaction concentration, the chromium(V) intermediate formed an EPR silent dimer, which dissociated upon dilution to lower concentration. UV/ visible experiments at pH 3.8 demonstrated that manganese(II) catalyzed the disproportionation of chromium(IV) to chromium(V) and chromium(III). The ability of the reaction intermediates to induce strand breaks in pBR322 DNA was determined at pH 3.8 and pH 5.8. At pH 3.8, chromium(IV) appeared to be the major species responsible for induction of strand breaks because the time course for formation of strand breaks did not parallel that of chromium(V), and strand breaks were decreased in the presence of the chromium(IV) scavenger manganese(II). At pH 5.8, fewer strand breaks were observed; however, the time course for their formation followed that of chromium(V). There has been much effort devoted to identification of the intermediate responsible for the induction of strand breaks during reactions of chromium(VI) with biological reductants. The current results suggest that it is not a single type of species that universally produces the DNA strand breaks observed in different chromium(VI) systems and that the reactivity of intermediates will depend on the chosen experimental conditions. Understanding this variability in chromium(VI) reactions may help to resolve the conflicting results from in vitro studies that are aimed at deciphering mechanisms of chromium(VI)-induced cancers.

Chromium↗
PubMed1996

Mapping conceptual to spatial relations in visual reasoning.

In 3 experiments, the authors investigated the impact of goals and perceptual relations on graph interpretation when people evaluate functional dependencies between continuous variables. Participants made inferences about the relative rate of 2 continuous linear variables (altitude and temperature). The authors varied the assignments of variables to axes, the perceived cause-effect relation between the variables, and the causal status of the variable being queried. The most striking finding was that accuracy was greater when the slope-mapping constraint was honored, which requires that the variable being queried be assigned to the vertical axis, so that steeper lines map to faster changes in the queried variable. The authors propose that graphs provide external instantiations of intermediate mental representations, enabling people to move from visuospatial representations to abstractions through the use of natural mappings between perceptual and conceptual relations.

Causality↗
PubMed2001

Nuclear factor-kappa B binding to the HIV-1 LTR in kidney: implications for HIV-associated nephropathy.

BACKGROUND: We have recently shown that renal epithelium is infected by HIV-1 and supports HIV-1 transcription in seropositive patients with renal disease. To investigate the regulation of HIV-1 gene expression in kidney, an HIV-1 transgenic mouse model was used to analyze the host transcriptional proteins that bind the 5' long-terminal repeat (LTR). METHODS: Viral gene expression was assessed in transgenic mouse tissue using Northern blotting and mRNA in situ hybridization. The transcription factors involved in LTR binding were determined using electrophoretic mobility shift assays. Cytoplasmic and nuclear extracts were prepared from tissues with varied levels of transgene expression. The binding of transcription factors to specific LTR fragments was determined using DNA competition experiments and supershifts with transcription factor-specific antibodies. RESULTS: Tissue-specific expression of the transgene was variable, with viral gene expression in the kidney at an intermediate level as compared with other tissues. Overall, the level of transgene expression directly correlated with abundance of nuclear factor-kappa B (NF-kappa B) in the nuclear extracts. High expressing tissue, however, had a constitutively active form of NF-kappa B. In contrast, the kidney contained an inducible NF-kappa B, which bound the LTR in combination with Sp1, suggesting a requirement for an activating event in renal HIV-1 expression of the LTR. CONCLUSIONS: These studies indicate that the regulation of the HIV-1 LTR in the kidney is similar to lymphoid tissues, and may explain, in part, why the HIV-1 life cycle is supported in kidney.

AIDS-Associated Nephropathy↗
PubMed2002

Tophaceous gout of the spine: MR imaging features.

AIM: To define the magnetic resonance (MR) imaging features of tophaceous gout of the spine. MATERIALS AND METHODS: We present the MR imaging examinations of 4 patients with spinal tophaceous gout. Spin-echo T1-weighted and fast spin-echo T2-weighted images were obtained for all patients, and 2 patients had gadolinium-enhanced MR imaging studies. Corresponding computed tomography (CT) was performed in one patient. All images were evaluated for the characteristics of the gouty tophi. RESULTS: The gouty tophi were located at the lower thoracic (n=1) and lumbar (n=3) levels. All tophi yielded homogeneous intermediate to low signal on T1-weighted images and variable signal intensity on T2-weighted images, comprising small foci of very low signal intensity on all sequences. Gadolinium-enhanced MR imaging studies revealed homogeneous enhancement or heterogeneous peripheral enhancement. Diffuse stippled calcifications were found in the tophi on CT images. Periarticular tophi with juxtaarticular bony erosions around facet joints occurred in 3 patients. CONCLUSION: Spinal tophaceous gout should be considered in the differential diagnosis when periarticular deposits contain very low signal foci on all MR imaging sequences.

Adult↗
PubMed2000

Tolerance to ischemia and hypoxia is reduced in aged human myocardium.

BACKGROUND: Recovery of cardiac function after cardiac surgery and other interventional cardiac procedures in elderly patients is inferior to that in younger patients, suggesting that the aged myocardium is more sensitive to ischemia and other stresses. Although convincing data from animal studies of senescence now exist, there is a dearth of controlled in vitro studies that examine the specific response of aged human myocardium to the stress of hypoxia or ischemia. OBJECTIVE: We sought to determine the effect of age on the capacity of human atrial trabeculae to recover contractile function after in vitro hypoxic or ischemic stress. METHODS: Atrial pectinate trabeculae were dissected from the tip of 58 right atrial appendages harvested during an operation in patients aged between 34 and 89 years and electrically stimulated at 1 Hz in oxygenated Ringer's solution at 37 degrees C. Tissues experienced 30 minutes of either hypoxia (N(2) and perfusate) or simulated ischemia (humidified N(2) without perfusate) and were returned to normoxia for recovery of function for 30 minutes. Developed force and other contractile variables were determined during each period. RESULTS: Under normoxic conditions, no significant age difference was observed for any contractile function variable. However, after hypoxia, the old (70-89 years) and intermediate age groups (60-69 years) showed reduced recovery of developed force (48.5% +/- 22.2% [n = 11] and 44.9% +/- 19% [n = 12], respectively) compared with that found (66.4% +/- 19.7% [n = 15]) in the younger (34-59 years) group (mean +/- SD, P =.02). Similarly, after simulated ischemia, the groups of 70- to 89-year-old and 60- to 69-year-old subjects showed reduced recovery of developed force (35.7% +/- 17% [n = 5] and 51.1% +/- 11.8% [n = 9], respectively) compared with that found (68.2% +/- 10.4% [n = 6]) in the group of 34- to 59-year-old subjects (P =.01). Multivariable analysis, comparing 20 factors of surgical patient characteristics and recovery of developed force, found that only age (P =.01) and hypertension (P =.01) were predictors of reduced recovery of developed force after either hypoxia or simulated ischemia. CONCLUSIONS: In aged human atrial myocardium, the capacity to recover contractile function after in vitro hypoxia or simulated ischemia is reduced compared with the younger myocardium of mature adults. These findings suggest that enhanced myocardial protective strategies may be indicated for elderly patients undergoing cardiac surgery.

Adult↗
PubMed1986

Characterization of beta-lactamases in clinical isolates of Bacteroides.

A total of 78 strains of Bacteroides was freshly isolated from clinical specimens; 60 of the strains were identified as B. fragilis and 16 as B. thetaiotaomicron. Overall, imipenem and the combination of benzylpenicillin and clavulanic acid were found to be considerably more active than cefoxitin or latamoxef (moxalactam) against these strains. Nearly all the strains exhibited weak beta-lactamase activity and 13 were judged to produce elevated levels of enzyme. These 13 strains fell into three groups: four strains that produced enzymes of low specific activity which were very susceptible to beta-lactamase inhibitors; three strains that produced enzymes of intermediate specific activity which hydrolysed cefoxitin and latamoxef at a variable rate; two of these enzymes rapidly inactivated imipenem and were insusceptible to beta-lactamase inhibitors; six strains that produced enzymes of high specific activity which were susceptible to beta-lactamase inhibitors; these strains did not inactivate beta-lactamase-stable beta-lactam antibiotics, but two of the strains nonetheless exhibited reduced susceptibility to latamoxef and imipenem.

Anti-Bacterial Agents↗
PubMed1985

Validation and use of resource utilization groups as a case-mix measure for long-term care.

A companion article describes the development of a patient classification system for long-term care patients, Resource Utilization Groups (RUGs). Three potential limitations of this system and its development are addressed here: the use of a subjectively determined dependent variable, geographic limitation of the data to Connecticut skilled nursing facilities, and limited assessment of the quality of the facilities studied. Additional systems of Resource Utilization Groups were derived, using the same clustering technique but employing two separate data sets from the Battelle Human Affairs Research Center. These data bases provided an objective dependent variable, wide geographic distribution of both skilled nursing facilities and intermediate care facilities, and homes specifically selected on the basis of quality. The RUGs derived from the two sets of Battelle data and the initial RUG system showed remarkable similarity in their patient groupings and in the case-mix indexes developed for nursing homes. The concurrence of the results obtained for these three systems greatly strengthens the basis for the use of this classification system as a case mix measure for long-term care.

Activities of Daily Living↗
PubMed2001

Early graft function after pediatric liver transplantation: comparison between in situ split liver grafts and living-related liver grafts.

BACKGROUND: The systematic application of living-related and cadaveric, in situ split-liver transplantation has helped to alleviate the critical shortage of suitable-sized, pediatric donors. Undoubtedly, both techniques are beneficial and advantageous; however, the superiority of either graft source has not been demonstrated directly. Because of the potential living-donor risks, we reserve the living donor as the last graft option for pediatric recipients awaiting liver transplantation. Inasmuch as no direct comparison between these two graft types has been performed, we sought to perform a comparative analysis of the functional outcomes of left lateral segmental grafts procured from these donor sources to determine whether differences do exist. METHODS: A retrospective analysis of all liver transplants performed at a single institution between February 1984 and January 1999 was undertaken. Only pediatric (<18 years) recipients of left lateral segmental grafts procured from either living-related (LRD) or cadaveric, in situ split-liver (SLD) donors were included. A detailed analysis of preoperative, intraoperative, and postoperative variables was undertaken. Survival was estimated using the Kaplan-Meier method, and comparison of variables between groups was undertaken using the t test of Wilcoxon rank sum test. RESULTS: There were no significant differences in the preoperative variables between the 39 recipients of SLD grafts and 34 recipients of LRD grafts. The donors did differ significantly in mean age, ABO blood group matching, and preoperative liver function testing. Postoperative liver function testing revealed significant early differences in aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, prothrombin time, and alkaline phosphatase, with grafts from LRD performing better than those from SLD. SLD grafts also had significantly longer ischemia times and a higher incidence of graft loss owing to primary nonfunction and technical complications (9 vs. 2, P<0.05). However, six of these graft losses in the SLD group were because of technical or immunologic causes, which, theoretically, should not differ between the two groups. Furthermore, these graft losses did not negatively impact early patient survival as most patients were successfully rescued with retransplantation (30-day actuarial survival, 97.1% SLD vs. 94.1% LRD, P=0.745). In the surviving grafts, the early differences in liver function variables normalized. CONCLUSIONS: Inherent differences in both donor sources exist and account for differences seen in preoperative and intraoperative variables. Segmental grafts from LRD clearly performed better in the first week after transplantation as demonstrated by lower liver function variables and less graft loss to primary nonfunction. However, the intermediate function (7-30 days) of both grafts did not differ, and the early graft losses did not translate into patient death. Although minimal living-donor morbidity was seen in this series, the use of this donor type still carries a finite risk. We therefore will continue to use SLD as the primary graft source for pediatric patients awaiting liver transplantation.

Adult↗
PubMed2005

The impact of thiopurine s-methyltransferase polymorphism on azathioprine-induced myelotoxicity in renal transplant recipients.

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. TPMT activity exhibits an interindividual variability, mainly as a result of genetic polymorphism. Patients with intermediate or deficient TMPT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. It has previously been reported that 3 variant alleles: TPMT*2, *3A, and *3C are responsible for over 95% cases of low enzyme activity. The purpose of this study was to explore the association between these polymorphisms and the occurrence of azathioprine adverse effects in 112 renal transplant recipients undergoing triple immunosuppressive therapy including azathioprine, cyclosporine, and prednisone. TPMT genetic polymorphism was determined using PCR-RFLP and allele-specific PCR methods. Azathioprine dose, leukocyte, erythrocyte, and platelet counts, graft rejection episodes, as well as cyclosporine levels were analyzed throughout the first year after organ transplantation. We found the frequency of leukopenia episodes (WBC < 4.0 x 10(9)/L) significantly higher in heterozygous patients (53.8%) compared with those with TPMT wild-type genotype (23.5%). One patient, who was a compound homozygote (3A/*3C), experienced severe azathioprine-related myelotoxicity each time after receiving the standard drug dose. Our results suggest that polymorphisms in TPMT gene may be responsible for approximately 12.5% of all leukopenia episodes in renal transplant recipients treated with azathioprine. Genotyping for the major TPMT variant alleles may be a valuable tool in preventing AZA toxicity and optimization of immunosuppressive therapy.

Adolescent↗
PubMed1990

Towards an understanding of the structural basis of 'forbidden' transport pathways in the Escherichia coli lactose carrier: mutations probing the energy barriers to uncoupled transport.

Recent progress in the analysis of mutants of the Escherichia coli lactose carrier function is reviewed, with special emphasis on the structural basis for energy barriers which prevent 'forbidden' conformational changes. Mutations which break down the barriers to forbidden isomerizations involving the binary carrier:sugar (CS) and carrier:proton (CH) complexes have been obtained in several laboratories. These mutants allow uncoupled transport of H+ or galactoside in the lactose carrier which normally couples cation and sugar movement in a 1:1 stoichiometry. These uncoupled mutants appear to be associated with changes in both sugar and cation recognition, suggesting that the physical interactions forming the basis for co-substrate recognition and uncoupling are not independently variable. By postulating that translocation involves transformation of the stable intermediate of the co-transport cycle to unstable transition state conformations of the carrier, it is possible to consider the consequences of mutagenesis in terms of transition state theory. Consistent with several experimental observations, the analysis predicts in each mutant the occurrence of more than one abnormality in the transport cycle (such as changes in sugar recognition, cation recognition or the coupling reaction). We have called the general phenomenon a 'mutational double-effect' because any mutation which alters the Gibbs free energy change of one reaction in the transport cycle must affect the free energy change of at least one other reaction in this cycle.

Biological Transport↗