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Competing Interests: Declaration of Competing Interest Heiko Yang: TL1DK139565, U2CDK133488; the remaining authors declare that they have no conflict of interest.

Objective: To describe the yield and indications for performing genetic testing in patients with nephrolithiasis. Genetic testing for nephrolithiasis is becoming more accessible and rapid due to technologic advances. This study aimed to determine the diagnostic yield of genetic testing in a cohort of high-risk stone formers and to identify 24-hour urine characteristics to prompt genetic screening., Materials and Methods: We retrospectively identified patients who underwent genetic testing for nephrolithiasis from 2020 to 22 at a single institution using a custom PerkinElmer genomics panel for nephrolithiasis. We compared characteristics of patients with and without genetic abnormalities. We used receiver operator characteristic (ROC) analysis to identify candidate thresholds for genetic testing., Results: Fourteen of 36 patients (39%) who underwent genetic testing had identifiable mutations. Five patients (14%) had known pathogenic mutations, including genes for primary hyperoxaluria (PH2 and PH3), cystinuria, and enamel-renal syndrome. The remaining mutations were variants of uncertain significance. Of the 14 patients with identified mutations, only 6 had concordant 24-hour urine abnormalities, including 3/5 with known pathogenic mutations. In patients with urine oxalate ≥ 40 mg/day, 3/29 (10.3%) had PH2 or PH3. ROC analysis showed that an oxalate threshold of ≥ 80 mg/day may have promising screening characteristics., Conclusion: Genetic testing for nephrolithiasis remains controversial due to unknown yield and the time and energy required to discuss results with patients. This preliminary report describes the yield and identifies clinical factors and a potential cut-off that may assist clinicians in deciding when genetic profiling should be pursued., Competing Interests: Declaration of Competing Interest Heiko Yang sources of funding: TL1DK139565, U2CDK133488; the remaining authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Objective: To determine whether early versus delayed autotransplantation are associated with adverse outcomes in patients undergoing renal autotransplantation., Methods: Patients who underwent renal autotransplantation from June 2012 to September 2022 were divided into 2 groups based on timing of autotransplant in relation to initial intervention or diagnosis (early cohort: ≤1-year; delayed cohort: >1-year). Primary outcomes were perioperative complications, aborted surgery, renal function (glomerular filtration rate [GFR]), and postoperative complications at most recent follow-up., Results: Autotransplantation patients (N = 72) were predominantly female (68%) and White (54%), with a median age of 49 years. Ninety percentage of patients had undergone previous interventions, including stenting (40%) and nephrostomy tubes (49%), primarily for obstruction (64%). Early versus delayed cohorts had median preoperative disease durations of 143 (IQR 83-222) versus 673 days (IQR 529-1703, P <.001), with similar median follow-up times (879 vs 818 days, P = .8). Groups were similar in demographics and comorbidities. There were no significant differences in rates of aborted surgery (15% vs 4.2%, P = .3), perioperative complications (15% vs 17%, P > .9), long-term complications (49% vs 48%, P > .9), or changes in GFR (median change +3 vs +4, P = .7). Outcomes were comparable across preoperative disease durations ranging from 6 to 24 months. These findings were confirmed following adjustments for sex, body mass index, American Society of Anesthesiologists classification, race, preoperative creatinine levels, laterality, gastroesophageal reflux disease, diabetes, hypertension, nephrolithiasis, hyperlipidemia, history of colon surgery, urologic surgery, abdominal surgery, and prior interventions in separate logistic models., Conclusion: Disease duration before autotransplantation does not influence outcomes, offering reassurance for clinical decision-making in complex cases., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Introduction: Patient perceptions of physician reimbursement commonly differ from actual reimbursement. This study aims to improve health care cost transparency and trust between patients, physicians, and the health care system by evaluating patient perceptions of Medicare reimbursement for artificial urinary sphincter (AUS) placement., Methods: We identified patients who underwent AUS placement at a single institution from 2014 to 2023. After obtaining informed consent, we administered a telephone survey to ask patients about their perceptions of Medicare reimbursement for AUS surgery and the amount they felt the physician should be compensated., Results: Sixty-four patients were enrolled and completed the survey. On average, patients estimated Medicare physician reimbursement to be $18,920, 25 times the actual average procedure reimbursement. Once informed that the actual amount was $757.52, 97% of respondents felt that the reimbursement was "somewhat lower" (13%) or "much lower" (84%) than what they considered fair. The average amount that patients felt the physician should be paid was $8,844, 12 times the actual average procedure reimbursement. Fifty-four percent of patients estimated their physician's reimbursement to be higher than what they later reported as being "fair," representing a presurvey belief that their physician was overpaid., Conclusions: Patient perceptions of physician reimbursement for AUS are vastly different than the actual amount paid. The discordance between patient perception and actual reimbursement could impact how patients view health care costs and the relationship with their provider.

Purpose: We evaluate the efficacy and safety profiles of currently available conservative management options for penile and urethral lichen sclerosus., Materials and Methods: A systematic review of existing literature on lichen sclerosus was conducted utilizing the PubMed, Embase, and Web of Science databases. References were assessed for relevance to nonsurgical management of male genital lichen sclerosus by title and abstract by 3 independent reviewers, then reviewed in full and in duplicate by 5 independent reviewers., Results: Seventeen studies describing conservative management of histologically confirmed penile and urethral lichen sclerosus in male patients were included in the final review. We present available evidence supporting the use of 4 major treatment modalities represented in the existing literature: topical corticosteroids, tacrolimus, platelet-rich plasma, and CO 2 laser. We also briefly discuss the limited studies on the use of oral acitretin and polydeoxyribonucleotide injections. Outcomes assessed include symptoms, clinical appearance, quality of life, sexual satisfaction, adverse effects, and long-term efficacy of treatment., Conclusions: Topical corticosteroids remain the mainstay of conservative management of penile and urethral lichen sclerosus, with current literature supporting the use of other therapies such as tacrolimus and platelet-rich plasma as alternatives or adjuvant treatments when escalation of treatment is necessary. Future research should further explore the efficacy and safety of newer therapies through additional controlled clinical trials in the targeted population.

Background: Ankle fractures are among the most common physeal fractures in children. When surgical management is warranted, subsequent hardware removal remains controversial. This study was designed to determine rates of hardware removal in patients with physeal ankle fractures and identify risk factors for removal. Procedure data was utilized to compare rates of subsequent ankle procedures in patients with hardware removed and hardware retained., Methods: We conducted a retrospective cohort study utilizing data from the Pediatric Health Information System (PHIS) for the years 2015 through 2021. We longitudinally followed patients treated for distal tibia physeal fractures to determine the rates of hardware removal and subsequent ankle procedures. Patients with open fractures or polytrauma were excluded. We used univariate, multivariate, and descriptive statistics to characterize the rates of hardware removal, identify factors associated with removal, and assess the rates of subsequent procedures., Results: This study included 1008 patients who underwent surgical management of a physeal ankle fracture. The mean age at index surgery was 12.6 years with an SD of 2.2 years, and 60% of patients identified as male. Two hundred forty-two patients (24%) had their hardware removed at an average time of 276 days (range, 21-1435 days) following index surgery. Patients with Salter Harris III (SH-III) or Salter Harris IV (SH-IV) fractures had hardware removed more often than patients with Salter-Harris II (SH-II) fractures (28.9% vs 11.7%, P  < .01). Four-year rates of subsequent ankle procedures are similar between patients with hardware removed and hardware retained., Conclusion: The rate of hardware removal in children with physeal ankle fractures is higher than previously reported. Patients of younger age, higher income, and with fractures involving the epiphysis (SH-III and SH-IV) are more likely to undergo hardware removal., Level of Evidence: Level III, retrospective study., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. ICMJE forms for all authors are available online., (© The Author(s) 2023.)

Background: Intermittent preventive treatment in infants (IPTi) is the administration of sulfadoxine-pyrimethamine (SP) at 2, 3, and 9 months of age to prevent malaria. We investigated the influence of IPTi on drug resistance., Methods: Twenty-four areas were randomly assigned to receive or not receive IPTi. Blood collected during representative household surveys at baseline and 15 and 27 months after implementation was tested for SP and resistance markers., Results: The frequency of SP in blood was similar in the IPTi and comparison areas at baseline and at 15 months. dhfr and dhps mutations were also similar at baseline and then increased similarly in both arms after 15 months of SP-IPTi. First-line treatment was switched from SP to artemether-lumefantrine before the final survey, when SP positivity fell among infants in comparison areas but increased in IPTi areas. This was accompanied by an increase in dhfr but not dhps mutations in IPTi areas (P = .004 and P = .18, respectively)., Conclusions: IPTi did not increase drug pressure or the selection on dhfr and dhps mutants, when SP was the first-line malaria treatment. Introduction of artemether-lumefantrine was followed by an increase in dhfr mutations, consistent with weak selection attributable to SP-IPTi, but not by an increase in dhps mutations, suggesting a fitness cost of this mutation.

Background: Sulphadoxine-pyrimethamine (SP) resistance is now widespread throughout east and southern Africa and artemisinin compounds in combination with synthetic drugs (ACT) are recommended as replacement treatments by the World Health Organization (WHO). As well as high cure rates, ACT has been shown to slow the development of resistance to the partner drug in areas of low to moderate transmission. This study looked for evidence of protection of the partner drug in a high transmission African context. The evaluation was part of large combination therapy pilot implementation programme in Tanzania, the Interdisciplinary Monitoring Programme for Antimalarial Combination Therapy (IMPACT-TZ) METHODS: The growth of resistant dhfr in a parasite population where SP Monotherapy was the first-line treatment was measured for four years (2002-2006), and compared with the development of resistant dhfr in a neighbouring population where SP + artesunate (SP+AS) was used as the first-line treatment during the same interval. The effect of the differing treatment regimes on the emergence of resistance was addressed in three ways. First, by looking at the rate of increase in frequency of pre-existing mutant dhfr alleles under monotherapy and combination therapy. Second, by examining whether de-novo mutant alleles emerged under either treatment. Finally, by measuring diversity at three dhfr flanking microsatellite loci upstream of the dhfr gene., Results: The reduction in SP selection pressure resulting from the adoption of ACT slowed the rate of increase in the frequency of the triple mutant resistant dhfr allele. Comparing between the two populations, the higher levels of genetic diversity in sequence flanking the dhfr triple mutant allele in the population where the ACT regimen had been used indicates the reduction in SP selection pressure arising from combination therapy., Conclusion: The study demonstrated that, alleles containing two mutations at the dhfr have arisen at least four times independently while those containing triple mutant dhfr arose only once, and were found carrying a single unique Asian-type flanking sequence, which apparently drives the spread of pyrimethamine resistance associated dhfr alleles in east Africa. SP+AS is not recommended for use in areas where SP cure rates are less than 80% but this study reports an observed principle of combination protection from an area where pyrimethamine resistance was already high.

The therapeutic efficacy of sulfadoxine-pyrimethamine (SP) in treating uncomplicated Plasmodium falciparum malaria is unevenly distributed in Colombia. The Andes mountain range separates regions in the west where malaria is endemic from those in the east and constitutes a barrier against gene flow and the dispersal of parasite populations. The distribution of dhfr and dhps genotypes of 146 P. falciparum samples from the eastern Amazon and Orinoco basins and Northwest and Southwest Pacific regions of Colombia was consistent with the documented levels of therapeutic efficacy of SP. The diversity of four dhfr- and dhps-linked microsatellites indicated that double- and triple-mutant alleles for both resistance loci have a single origin. Likewise, multilocus association genotypes, including two unlinked microsatellite loci, suggested that genetic exchanges between the eastern Orinoco and Northwest Pacific populations has taken place across the Andes, most probably via migration of infected people.

Background: It is argued that, the efficacy of anti-malarials could be prolonged through policy-mediated reductions in drug pressure, but gathering evidence of the relationship between policy, treatment practice, drug pressure and the evolution of resistance in the field is challenging. Mathematical models indicate that drug coverage is the primary determinant of drug pressure and the driving force behind the evolution of drug resistance. These models show that where the basis of resistance is multigenic, the effects of selection can be moderated by high recombination rates, which disrupt the associations between co-selected resistance genes., Methods: To test these predictions, dhfr and dhps frequency changes were measured during 2000-2001 while SP was the second-line treatment and contrasted these with changes during 2001-2002 when SP was used for first-line therapy. Annual cross sectional community surveys carried out before, during and after the policy switch in 2001 were used to collect samples. Genetic analysis of SP resistance genes was carried out on 4,950 Plasmodium falciparum infections and the selection pressure under the two policies compared., Results: The influence of policy on the parasite reservoir was profound. The frequency of dhfr and dhps resistance alleles did not change significantly while SP was the recommended second-line treatment, but highly significant changes occurred during the subsequent year after the switch to first line SP. The frequency of the triple mutant dhfr (N51I,C59R,S108N) allele (conferring pyrimethamine resistance) increased by 37% - 63% and the frequency of the double A437G, K540E mutant dhps allele (conferring sulphadoxine resistance) increased 200%-300%. A strong association between these unlinked alleles also emerged, confirming that they are co-selected by SP., Conclusion: The national policy change brought about a shift in treatment practice and the resulting increase in coverage had a substantial impact on drug pressure. The selection applied by first-line use is strong enough to overcome recombination pressure and create significant linkage disequilibrium between the unlinked genetic determinants of pyrimethamine and sulphadoxine resistance, showing that recombination is no barrier to the emergence of resistance to combination treatments when they are used as the first-line malaria therapy.

Objectives: To assess the extent of drug resistance in Uige through molecular genetic analysis and to test whether the dhfr triple mutant alleles present in Angola are of southeast Asian origin., Methods: Seventy-one samples of blood from children admitted to the Pediatric Emergency Unit of Uige Provincial Hospital in 2004 were screened for resistance mutations at pfcrt, pfmdr1, pfdhfr, pfdhps and pfATPase6., Results: Mutations in pfcrt (codon76), pfmdr1 (codon86), pfdhfr (codons 51, 59, 108) and pfdhps (codons 436, 437) were common. Among the 66 isolates for which we were able to determine complete genetic information 13.7% carried all seven of these mutations. Flanking microsatellite analysis revealed the triple mutant pfdhfr was derived from the southeast Asian lineage, while the N51I+S108N double mutant pfdhfr alleles are a local origin. pfATPase6 mutations were rare and S769N was not found., Conclusion: The parasite population of Uige Angola has high frequency mutations in pfcrt, dhfr and dhps associated with resistance to chloroquine and sulphadoxine pyrimethamine, reflecting past reliance on these two drugs which were the mainstay of treatment until recently. Our findings show that drug resistance in Uige has occurred through a combination of local drug pressure and the regional and international dispersal of resistance mutant alleles.

Treatment of acute malaria caused by Plasmodium falciparum may include long-half-life drugs, such as the antifolate combination sulfadoxine-pyrimethamine (SP), to provide posttreatment chemoprophylaxis against parasite recrudescence or delayed emergence from the liver. An unusual case of P. falciparum recrudescence in a returned British traveler who received such a regimen, as well as a series of 44 parasite isolates from the same hospital, was analyzed by PCR and direct DNA sequencing for the presence of markers of parasite resistance to chloroquine and antifolates. The index patient harbored a mixture of wild-type and resistant pfdhfr and pfdhps alleles upon initial presentation. During his second malaria episode, he harbored only resistant parasites, with the haplotypes IRNI (codons 51, 59, 108, and 164) and SGEAA (codons 436, 437, 540, 581, and 613) at these two loci, respectively. Analysis of isolates from 44 other patients showed that the pfdhfr haplotype IRNI was common (found in 81% of cases). The SGEAA haplotype of pfdhps was uncommon (found only in eight cases of East African origin [17%]). A previously undescribed mutation, I431V, was observed for seven cases of Nigerian origin, occurring as one of two haplotypes, VAGKGS or VAGKAA. The presence of this mutation was also confirmed in isolates of Nigerian origin from the United Kingdom Malaria Reference Laboratory. The presence of the pfdhps haplotype SGEAA in P. falciparum parasites of East African origin appears to compromise the efficacy of treatment regimens that include SP as a means to prevent recrudescence. Parasites with novel pfdhps haplotypes are circulating in West Africa. The response of these parasites to chemotherapy needs to be evaluated.

Background: Although the molecular basis of resistance to a number of common antimalarial drugs is well known, a geographic description of the emergence and dispersal of resistance mutations across Africa has not been attempted. To that end we have characterised the evolutionary origins of antifolate resistance mutations in the dihydropteroate synthase (dhps) gene and mapped their contemporary distribution., Methods and Findings: We used microsatellite polymorphism flanking the dhps gene to determine which resistance alleles shared common ancestry and found five major lineages each of which had a unique geographical distribution. The extent to which allelic lineages were shared among 20 African Plasmodium falciparum populations revealed five major geographical groupings. Resistance lineages were common to all sites within these regions. The most marked differentiation was between east and west African P. falciparum, in which resistance alleles were not only of different ancestry but also carried different resistance mutations., Conclusions: Resistant dhps has emerged independently in multiple sites in Africa during the past 10-20 years. Our data show the molecular basis of resistance differs between east and west Africa, which is likely to translate into differing antifolate sensitivity. We have also demonstrated that the dispersal patterns of resistance lineages give unique insights into recent parasite migration patterns.

The antimalarial combination of sulfadoxine and pyrimethamine (SP) was introduced as first-line treatment for uncomplicated malaria in Tanzania during 2001 following 18 years of second-line use. The genetic determinants of in vitro resistance to the two drugs individually are shown to be point mutations at seven sites in the dihydrofolate reductase gene (dhfr) conferring resistance to pyrimethamine and five sites in the dihydropteroate synthase (dhps) gene conferring resistance to sulfadoxine. Different combinations of mutations within each gene confer differing degrees of insensitivity, but information about the frequency with which allelic haplotypes occur has been lacking because of the complicating effects of multiple infection. Here we used a novel high-throughput sequence-specific oligonucleotide probe-based approach to examine the present resistance status of three Plasmodium falciparum populations in northern Tanzania. By using surveys of asymptomatic infections and screening for the presence of all known point mutations in dhfr and dhps genes, we showed that just five dhfr and three dhps allelic haplotypes are present. High frequencies of both triple-mutant dhfr and double-mutant dhps mutant alleles were found in addition to significant interregional heterogeneity in allele frequency. In vivo studies have shown that the cooccurrence of three dhfr mutations and two dhps mutations in an infection prior to treatment is statistically predictive of treatment failure. We have combined data for both loci to determine the frequency of two-locus genotypes. The triple-dhfr/double-dhps genotype is present in all three regions with frequencies ranging between 30 and 63%, indicating that treatment failure rates are likely to be high.

Human antibodies to the block 2 region of Plasmodium falciparum merozoite surface protein 1 (MSP1) are associated with a reduced prospective risk of clinical malaria. Block 2 is highly polymorphic, but all known alleles can be grouped into three major types. Two of these types (the K1-like and MAD20-like types) contain type-specific sequences (found in all alleles of a particular type) that flank polymorphic tripeptide repeats. These repeats contain both type-specific and subtype-specific sequences. To evaluate the antibody recognition of these parts of block 2, a new panel of six recombinant proteins was used (fused type-specific flanking sequences and two representative repeat sequences for each of the K1-like and MAD20-like types separately). Extensive testing of these antigens and full-length block 2 antigens showed that human serum immunoglobulin G antibodies induced by infection can recognize (i) type-specific epitopes in the repeats, (ii) subtype-specific epitopes in the repeats, or (iii) type-specific epitopes in flanking sequences. A large prospective study in The Gambia showed that antibodies to the repeats are strongly associated with protection from clinical malaria. The results are important for design of a vaccine to induce protective antibodies, and they address hypotheses about repeat sequences in malaria antigens.

Trisomy 16 (Ts16) mouse is considered an animal model of Down syndrome (human trisomy 21). Whole-cell patch-clamp was used to evaluate potassium and chloride currents of cultured tongue muscle cells from fetal Ts16 and diploid mice. No difference was found in membrane capacitance between the two groups. K(+) and Cl(-) currents were pharmacologically isolated. K(+) conductance was reduced by 31% in Ts16 cells (373 pS/pF) compared with diploid cells (539 pS/pF). Cl(-) conductance was 51% larger in Ts16 cells (103 pS/pF) compared with diploid cells (68 pS/pF). However kinetics for K(+) and Cl(-) currents did not differ between the cell types. An increase in Cl(-) conductance and a decrease in K(+) conductance in Ts16 muscle cells, if present in muscle of Down syndrome subjects, might account for the observed hypotonia in these subjects.

The extent of the early stage of the Maillard-type reaction that impaired functional properties of whey proteins was evaluated by electrospray ionization mass spectrometry. Under conditions of mild heat treatment (63 degrees C for 20 s) applied to milk before whey separation at room temperature 23 degrees C), a modification of the relative molecular mass of beta-lactoglobulin (beta-LG) was observed that differed from that of the native form by 324. This specific modification of beta-LG occurred in acidified whey as well as in sweet whey and increased with the extent of the heat treatment. Incubation of purified beta-LG dissolved in milk ultrafiltration permeate or in lactose solution at 50 to 80 degrees C demonstrated the presence of a lactosyl residue that was covalently bound to beta-LG; beta-casein, used as a control, showed no mass modification. Studies of kinetics showed that a maximum of 35% of the beta-LG was lactosyl-beta-LG conjugate after heat treatment at 70 degrees C for 1 h. This study provides the first direct evidence of specific lactosylation of beta-LG during the initial stage of the Maillard reaction. One of the first lactose-binding sites was identified as a Lys47 by protease mapping and analysis by means of on-line liquid chromatography combined with mass spectrometry. In addition, collision-activated dissociation performed on the lactosylated peptide beta-LG (f 46-51) showed the rearrangement reactions occurring during the fragmentation process by electrospray. A mechanism is proposed.

Electrospray ionization (ESI) was used to adapt the thermospray ionization liquid chromatographic/mass spectrometric (LC/MS) method of Liberato et al. to the determination of acetylcholine and choline released from cells in culture. A clonal cell line derived from a rat pheochromocytoma (PC12) was used and cultures depolarized in the presence of cholinesterase inhibitor to maximize the recovery of acetylcholine from the culture medium and protect against its hydrolysis. Concentration of acetylcholine and choline released into the incubation medium was accomplished with Sep-Pak C16 cartridges. The preformed analyte cations were resolved by reversed-phase high-performance liquid chromatography and analyzed without derivatization by electrospray ionization mass spectrometry with the use of acetyl[2H9]choline as internal standard. LC/MS allows for the direct determination of acetylcholine and choline, which is not possible with other methods.

Management of dairy whey has often involved implementation of the most economical disposal methods, including discharge into waterways and onto fields or simple processing into low value commodity powders. These methods have been, and continue to be, restricted by environmental regulations and the cyclical variations in price associated with commodity products. In any modern regimen for whey management, the focus must therefore be on maximizing the value of available whey solids through greater and more varied utilization of the whey components. The whey protein constituents offer tremendous opportunities. Although whey represents a rich source of proteins with diverse food properties for nutritional, biological, and functional applications, commercial exploitation of these proteins has not been widespread because of a restricted applications base, a lack of viable industrial technologies for protein fractionation, and inconsistency in product quality. These shortcomings are being addressed through the development of novel and commercially relevant whey processing technologies, the preparation of new whey protein fractions, and the exploitation of the properties of these fractions in food and in nontraditional applications. Examples include the following developments: 1) whey proteins as physiologically functional food ingredients, 2) alpha-lactalbumin and beta-lactoglobulin as nutritional and specialized physically functional food ingredients, and 3) minor protein components as specialized food ingredients and an important biotechnological reagents. Specific examples include the isolation and utilization of lactoferrin and the replacement of fetal bovine serum in tissue cell culture applications with a growth factor extract isolated from whey.

1. The convulsant barbiturate 5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid (CHEB) depolarizes dorsal root ganglion (DRG) neurons. We have applied microfluorimetric and whole-cell patch clamp techniques to investigate the mechanisms underlying this response in freshly dissociated mouse DRG cells. 2. Application of CHEB (2-200 microM) raised cytosolic calcium concentration ([Ca2+]i) rapidly and reversibly in 55% of eighty-three neurons tested. This population did not correlate with other classifications of sensory neurons based on either cell size or the expression of membrane currents. 3. The response was dependent on external calcium and was reduced by 81 +/- 22% by Ruthenium Red. A rise in [Ca2+]i was still seen with the membrane potential clamped at -70 mV, excluding membrane depolarization and activation of voltage-dependent Ca2+ channels as the principal mechanism for the response. 4. The rise in [Ca2+]i was associated with an increase in membrane conductance and a current, ICHEB, which was inward at -70 mV. Both the rise in [Ca2+]i and the current showed 'run-down' under whole-cell recording conditions. When K+ conductances were blocked, the reversal potential of ICHEB was close to 0 mV. This was independent of the Cl- reversal potential, suggesting that ICHEB is carried as a non-specific cation current. 5. In contrast to the change in [Ca2+]i, ICHEB was not dependent on external Ca2+ and the current was still seen when [Ca2+]i as strongly buffered by the pipette filling solution. These data suggest that CHEB opens a non-selective cation channel permeant to Ca2+, raising [Ca2+]i and further depolarizing the cell membrane potential. The exact nature of this conductance remains unknown. These actions could readily account for the convulsant actions of the drug, depolarizing neurons and increasing transmitter release. 6. It was also noted that CHEB increases autofluorescence derived from mitochondrial NAD(P)H. Further examination of this phenomenon using the dye rhodamine 123 to follow changes in mitochondrial potential (psi m) suggested that CHEB is a potent inhibitor of mitochondrial respiration, probably acting at complex I. These effects appeared to be quite distinct from the action of CHEB at the level of the plasma membrane.

The whole cell configuration of the patch clamp technique has been applied to identify the membrane currents expressed by populations of dissociated mouse primary sensory neurons. Three discrete populations of cells were distinguished on the basis of cell size and the array of currents expressed. Group 1 cells (capacitance 10-30 pF) expressed a Na+ current resistant to tetrodotoxin (1 microM) and a prominent, low threshold, inactivating, K+ current sensitive to 4-aminopyridine (IA). A population (53%) of these small cells responded to capsaicin (10 microM) with an inward current, suggesting a functional correlate with nociceptive "C"-cells. The cells of Group 2 (capacitance 55-85 pF) were characterized by the expression of a Na+ current sensitive to tetrodotoxin and a prominent inward current activated by hyperpolarization (IH). They also showed a variant of the A-type K+ current, which was a low threshold, but sustained K+ current, sensitive to dendrotoxin (30 nM). Group 3 cells, of intermediate size (capacitance 30-55 pF) were similar to Group 2 cells, in that they expressed a tetrodotoxin-sensitive Na+ current and (through reduced in amplitude), IH. The most notable feature of Group 3 cells was the expression of a transient, low threshold Ca2+ current. The differential expression of these conductances was reflected in the behaviour of cells under current clamp control. Each group of cells could thus be distinguished by the selective expression of specific ionic conductances which correlated clearly with cell size, suggesting a correlation with well recognised functional differentiation of sensory neurons. The selective expression of specific subsets of membrane channels may provide valuable markers in studying the developmental regulation of phenotype in this population of cells.

Correlations were identified between levels of the native whey proteins, beta-lactoglobulin and alpha-lactalbumin and the surface and total hydrophobicities of cheese whey in response to different heat treatments. Heat-induced changes in the native beta-lactoglobulin content and surface hydrophobicity of whey exhibited the most significant linear relationship while correlations between total hydrophobicity and the native proteins were less significant because of an atypical rise in the n-heptane-binding capacity of whey after high-temperature treatment. The content of native beta-lactoglobulin in whey was more sensitive to heating than the content of native alpha-lactalbumin, while heat-related changes in the total hydrophobicity of whey were generally greater than similar changes in surface hydrophobicity.

A novel series of nonpeptidic angiotensin II (AII) receptor antagonists is reported, derived from linkage of the biphenylcarboxylic acid or biphenylyltetrazole moiety found in previously described antagonists via a methyleneoxy chain to the 4-position of a 2-alkyl quinoline. When evaluated in an in vitro binding assay using a guinea pig adrenal membrane preparation, compounds in this series generally gave IC50 values in the range 0.01-1 microM. Structure-activity studies showed the quinoline nitrogen atom and a short alkyl chain at the quinoline 2-position to be essential for receptor binding. On intravenous administration in a normotensive rat model, the more potent compounds inhibited the AII-induced pressor response with ED50 values in the range 0.1-2.0 mg/kg. One of the compounds, 2-ethyl-4-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methoxy]quinoline (5g), demonstrated good oral activity in two rat models. At doses in the range 1-10 mg/kg in AII-infused, normotensive rats, the compound exhibited a dose-related inhibition of the pressor response with a good duration of action at the higher doses. In a renal hypertensive rat model, compound 5g showed a rapid and sustained lowering of blood pressure at a dose of 5 mg/kg. On the basis of its profile, this compound, designated ICI D8731, has been selected for clinical evaluation.

This paper describes the synthesis of a series of N-[2-(1-pyrrolidinyl)ethyl]acetamides 1, variously substituted at the carbon adjacent to the amide nitrogen (C1), and related analogues, together with their biological evaluation as opioid kappa agonists. In the first part of the study, the variants in N-acyl, N-alkyl, and amino functions were explored when the substituent at C1 was 1-methylethyl and the optimum was found to be exemplified by 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-(1-methylethyl)-2- (1-pyrrolidinyl)ethyl]acetamide (13). Subsequently, racemic or chiral amino acids were used to introduce other alkyl and aryl substituents at C1 of the ethyl linking moiety. A series of potent compounds, bearing substituted-aryl groups at C1, were discovered, typified by 2-(3,4-dichloro-phenyl)-N-methyl-N-[(1R,S)-1-(3-aminophenyl)-2-(1- pyrrolidinyl)ethyl]acetamide (48), which was 5-fold more active as the racemate than 13 in vitro and exhibited potent naloxone-reversible analgesic effects (ED50 = 0.04 mg/kg sc) in a mouse abdominal constriction model.

The allergenicity of cow's milk whey proteins, purified by high performance liquid chromatography (HPLC), was examined by the radio-allergosorbent test (RAST) against the sera of children immediately hypersensitive to milk. beta-lactoglobulin and alpha-lactalbumin bound specific IgE in the sera of 63% and 75% of these patients respectively. These allergens were tested for cross reactivity with each other by RAST inhibition. Both inhibited the binding of IgE, in the sera of allergic patients, to the other protein. Two possible determinant peptides, one from beta-lactoglobulin and one from alpha-lactalbumin, were selected by computer prediction of antigenic sites and synthesized by the fluorenylmethoxycarbonyl (FMOC)-polyamide method. The peptides were adsorbed to nitrocellulose discs and used in further RAST studies with sera from the allergic children. Both peptides bound specific IgE in the RAST assay.

A series of 1,2,4-triazolo[4,3-a]pyrazine derivatives with human renin inhibitory activity, which incorporate (1S,2S)-2-amino-1,3-dicyclohexyl-1-hydroxypropane, statine (Sta), and (3S,4S)-4-amino-5-cyclohexyl-3-hydroxy-pentanoic acid (ACHPA) transition-state mimetics, have been prepared. Structure-activity relationships for renin inhibitory activity in the series are consistent with the 2-[8-isobutyl-6-phenyl-1,2,4-triazolo[4,3-a]pyrazin-3-yl]-3-(3 pyridyl)propionic acid moiety 10b acting as a non-peptidic replacement for the P4-P2 (Pro-Phe-His) residues of the natural substrate angiotensinogen. Compounds 12m, 12o and 12q were potent inhibitors of partially purified human renin (IC50 values 1.7, 6.8, and 3.7 nM, respectively), and also effectively lowered blood pressure in anesthetized, sodium depleted marmosets following intravenous administration. On oral administration however, no blood pressure lowering activity could be detected, and absorption studies in bile duct cannulated rats indicate that this may be due primarily to poor oral absorption, rather than rapid biliary excretion. The reason for the observed poor oral activity is not clear, but it seems unlikely that poor aqueous solubility or metabolic instability to gut enzymes are rate-determining, and other factors such as high molecular weight may also be very important.

ICI 169,369 (2-(2-dimethylaminoethylthio-3-phenylquinoline hydrochloride) has been tested in vivo for its potency and selectivity as an antagonist at 5-HT2 and 5-HT1C receptors. It caused a 50% inhibition of 5-HTP-induced head twitches in mice and fenfluramine-induced hyperthermia in the rat at approximately 1 mg/kg following parenteral administration. Results showed that ICI 169,369 had good oral bioavailability, since in the fenfluramine test the oral and s.c. ID50 values were similar. ICI 169,369 was a selective antagonist of 5-HT-induced bronchoconstriction in the guinea-pig and 5-HT-induced pressor effects in the anaesthetised dog. In a series of other tests in vivo the compound was shown to be devoid of significant activity at alpha 1- and alpha 2-adrenoceptors, dopamine (D2), muscarinic (M1) and histamine (H1) receptors at 30-100 times its ID50 values used in the 5-HT tests. Thus, ICI 169,369 is a selective, orally active 5-HT2/5-HT1C antagonist that should prove useful in the analysis of the role of 5-HT in physiological and pathological states.

A series of novel 1'-methylxanthene-9-spiro-4'-piperidines has been prepared in the search for opiate analgesics with improved pharmacological properties. It has been found that introduction of a hydroxyl group into the 4-position of the xanthenespiropiperidine nucleus produces a potent mu-opiate agonist. The structure-activity relationship of the series has been explored by use of isosteric replacements of the phenolic hydroxyl group. Moreover, the effect of altering the conformation of the piperidine ring has been studied. It was interesting to note that, in compounds lacking the phenolic hydroxyl group, opiate activity could be produced by introduction of the (phenylamino)ethyl group instead of methyl at the 1'-position.

ICI 169,369 is a chemically novel 5-HT antagonist that has higher affinity for the 5-HT2 binding sites in rat cortex than it has for 5-HT1 sites (Ki 1.79 x 10(-8) and 1.58 x 10(-6) M, respectively). In isolated tissue preparations ICI 169,369 was shown to be a competitive antagonist of 5-HT on the rabbit aorta, pig coronary artery and rat caudal artery. In the latter preparation it had a similar pA2 value to ketanserin (pA2 8.18 +/- 0.5 and 8.42 +/- 0.06, respectively). Unlike ketanserin, which was inactive, ICI 169,369 was a non-surmountable antagonist at the rat stomach fundus 5-HT 'D' receptor, recently reclassified as 5-HTIC. It was inactive (greater than 10(-6) M) at the 5-HT3 receptors found in the isolated perfused rabbit heart and the myenteric plexus of the guinea-pig ileum. At receptors other than those for 5-HT (alpha 1, alpha 2, beta 1, beta 2, H1, H2 and muscarinic), ICI 169,369 was inactive at concentrations of either 10(-6) or 10(-5) M. Thus the profile of ICI 169,369 should make it useful in the analysis of the role of 5-HT in physiological and pathological states.

A series of 2-[(2-aminoethyl)thio]quinolines substituted at the 3-position with alkyl, aryl, or heteroaryl groups has been prepared in the search for novel and selective 5-HT2 antagonists. The affinity of the compounds for 5-HT1 receptor sites was measured by their ability to displace [3H]-5-HT from rat brain synaptosomes whereas the affinity for 5-HT2 receptor sites was measured by their ability to displace [3H]spiperone from synaptosomes prepared from rat brain cortex. The 5-HT2 antagonist properties of the compounds were measured in vivo by their antagonism of 5-hydroxytryptophan-induced head twitches in the mouse and by their antagonism of hyperthermia induced by fenfluramine (N-ethyl-alpha-methyl-m-(trifluoromethyl)phenethylamine hydrochloride) in the rat. The structure-activity relationships in this series are discussed and the properties of 2-[[2-(dimethylamino)ethyl]thio]-3-phenylquinoline hydrochloride (70) are highlighted.

Bovine milk lysozyme has been partially purified by a method developed in this laboratory. We have shown, by preliminary sequential analysis, and by gel filtration on HPLC, that the product is a mixture of two components. One of these, the enzymically active one, differs in its N-terminal sequence from that of "lysozyme 2", a bovine stomach mucosal enzyme, by 7 residues within the first 39 residues. However, some of its properties differ markedly from those of lysozyme 2. The other component, comprising 70% by weight of the total mixture, bears no sequential resemblance to any protein known to us. Our two component system appears to be the same as the preparation of Chandan et al. (Biochim. Biophys. Acta 110, 289 (1965], which they concluded was an homogeneous preparation of lysozyme.

Amino acids have been separated by gas-liquid chromatography as their N-heptafluorobutyryl isobutyl esters. Complete resolution of derivatives of all the common amino acids has been achieved using a high-performance support-coated open-tubular capillary column. The analysis time was 30 min. Modifications to the derivatization procedure of MacKenzie and Tenaschuk have been introduced. Acylation by heating at 150 degrees was shown to be destructive; 110 degrees has been selected for routine preparation. To obtain a volatile histidine derivative it has been found necessary to add an antioxidant and to heat samples with ethoxyformic anhydride prior to injection. Amino acid analysis of beta-lactoglobulin after 6 N HCl digestion yielded results in good agreement with those obtained by the conventional ion-exchange method. The method has also been successfully applied to estimation of the different caseins in whole casein and in purified fractions by amino acid analysis of residues liberated by carboxy-peptidase digestion.

Heat stability as a function of pH has been studied in concentrated and non-concentrated milk systems. The influence of preheating has also been examined. The concentration of beta-lactoglobulin has been shown to affect markedly the heat stability behaviour in both systems but with different characteristics. Increasing the level of urea resulted in increased heat stability in non-concentrated milks, but corresponding concentrated milks showed a small decrease in maximum heat stability. It has not been possible to extrapolate heat stability data from non-concentrated to concentrated milks.