Search

We describe the evolution of the nonprofit Nepal Ambulance Service (NAS) in a narrative of its 10-y history, presenting geographical, social, cultural, and financial considerations that permeated the development of NAS. We gathered narrative information from the NAS leadership and partners to detail key organizational considerations regarding the implementation and maintenance of the prehospital system in Nepal. We describe the response of NAS to the 2015 earthquake and summarize transport data for 6 mo before and 6 mo after the event. The data collected included the date and time of calls received, time to ambulance dispatch, on-scene time, time to arrival at the hospital, time until the ambulance crew was back in service, patient age and sex, chief complaints, and work shift time of the ambulance crew. To characterize the time to response and transport after the 2015 earthquake, we present the means and standard deviations of the time intervals. There was an overall increase in calls and, specifically, trauma-related calls after the 2015 earthquake. The time from a call placed to dispatch was stable, approximately 2 min, throughout the period, whereas the time from dispatch to the scene and arrival at the scene varied widely. We discuss the response to coronavirus disease 2019 (COVID-19). The NAS provided care to 1230 patients with COVID-19. The descriptive data show how well NAS responded to a major national disaster and the recent pandemic., (Copyright © 2022 Wilderness Medical Society. Published by Elsevier Inc. All rights reserved.)

Purpose: Several reports have shown that cells with p53 mutations display increased resistance to ionizing radiation, a treatment often used clinically for localized prostate carcinoma., Materials and Methods: Totals of 18 post-irradiated locally recurrent prostatic carcinoma specimens and 25 (no radiation) stage D1 node-positive (TxN+MO) primary prostatic carcinoma specimens were tested for p53 immunoreactivity by immunohistochemistry. Of the 18 post-radiation locally recurrent prostatic carcinomas 10 were further analyzed by single strand conformational polymorphism to assess the validity of using this immunohistochemistry approach in irradiated tissue for detecting p53 alterations. Specimens showing p53 alterations by single strand conformational polymorphism were subjected to nucleotide sequence analysis or tested for loss of heterozygosity at a locus within the p53 gene., Results: Of the 25 stage TxN+MO prostatic carcinomas without radiation 5 (20%) were immunoreactive (consistent with the reported incidence of positive immunoreactivity in clinical/surgical stage TxN+MO primary prostatic carcinomas). In contrast, 13 of 18 post-radiation locally recurrent prostatic carcinoma specimens (72%) were immunoreactive. Multivariate logistic regression analysis showed no dependence of p53 immunoreactivity to grade, stage or androgen status in the post-radiation locally recurrent prostatic carcinoma group, while 8 of 10 hormone naive prostatic carcinoma specimens (80%) were immunoreactive. The temporal relationship between p53 alterations and radiotherapy was assessed. Pre-irradiation prostatic carcinomas available from 5 patients with immunoreactive post-radiation locally recurrent disease were analyzed and all were immunoreactive., Conclusions: p53 Alteration in localized prostatic carcinoma is uncommon. Our study confirms others in that even aggressive locally advanced nonirradiated primaries (stage TxN+MO) contain only 20% incidence of p53 alterations. However, our study demonstrates that p53 alterations are found in the preponderant majority of post-radiation locally recurrent prostatic carcinoma specimens. Limited evaluation of pretreatment prostatic carcinoma biopsies uniformly documented the presence of p53 alterations before ionizing radiation, thereby demonstrating that p53 alteration was already present and was not radiation-induced or only correlated with late stage disease. This finding suggests a potential for p53 immunoreactivity to be used as a pretreatment marker that might predict local treatment failure with ionizing radiation. Large scale prospective trials would appear warranted to evaluate conclusively the potential prognostic applicability of p53 pre-screening before enrollment in definitive radiotherapy.

The genetic alterations associated with prostate carcinogenesis, its progression, and the emergence of androgen independence gradually are being defined. This article focuses on a number of genetic defects that have been identified in prostate cancer, specifically as they relate to disease progression, radiation resistance, and androgen independence.

The 2.3-A crystal structure of recombinant human dihydrofolate reductase (EC 1.5.1.3, DHFR) has been solved as a binary complex with folate (a poor substrate at neutral pH) and also as a binary complex with an inhibitor, 5-deazafolate. The inhibitor appears to be protonated at N8 on binding, whereas folate is not. Rotation of the peptide plane joining I7 and V8 from its position in the folate complex permits hydrogen bonding of 5-deazafolate's protonated N8 to the backbone carbonyl of I7, thus contributing to the enzyme's greater affinity for 5-deazafolate than for folate. In this respect it is likely that bound 5-deazafolate furnishes a model for 7,8-dihydrofolate binding and, in addition, resembles the transition state for folate reduction. A hypothetical transition-state model for folate reduction, generated by superposition of the DHFR binary complexes human.5-deazafolate and chicken liver.NADPH, reveals a 1-A overlap of the binding sites for folate's pteridine ring and the dihydronicotinamide ring of NADPH. It is proposed that this binding-site overlap accelerates the reduction of both folate and 7,8-dihydrofolate by simultaneously binding substrate and cofactor with a sub van der Waals separation that is optimal for hydride transfer.

The role of the active site residue phenylalanine-31 (Phe31) for recombinant human dihydrofolate reductase (rHDHFR) has been probed by comparing the kinetic behavior of wild-type enzyme (wt) with mutant in which Phe31 is replaced by leucine (F31L rHDHFR). At pH 7.65 the steady-state kcat is almost doubled, but the rate constant for hydride transfer is decreased to less than half that for wt enzyme, as is the rate of the obligatory isomerization of the substrate complex that precedes hydride transfer. Although steady-state measurements indicated that the mutation causes large increases in Km for both substrates, dissociation constants for many complexes are decreased. These apparent paradoxes are due to major mutation-induced decreases in rate constants (koff) for dissociation of folate, dihydrofolate, and tetrahydrofolate from all of their complexes. This results in a mechanism proceeding almost entirely by only one of the two pathways used by wt enzyme. Other consequences of these changes are a much altered dependence of steady-state kcat on pH, inhibition rather than activation by tetrahydrofolate, absence of hysteresis in transient-state kinetics, and a decrease in enzyme efficiency under physiological conditions. The results indicate that there is no quantitative correlation between dihydrofolate binding and the rate of hydride transfer for this enzyme.

Association and dissociation rate constants obtained by stopped-flow spectroscopy have permitted definition of a kinetic scheme for recombinant human dihydrofolate reductase that correctly predicts full time course kinetics of the enzymatic reaction over a wide range of substrate and product concentrations. The scheme is complex compared with that for the bacterial enzyme and involves branched pathways. It successfully accounts for observed rapid hysteresis preceding steady state and for the nonhyperbolic dependence of steady-state rate on substrate and product concentrations. The major branch point in the catalytic cycle occurs at E.NADP.H4folate because either NADP or H4folate can dissociate from the ternary product complex (koff = 84 s-1 and 46 s-1, respectively). The rate of conversion of enzyme-bound substrates to products is very fast (k = 1360 s-1) and nearly unidirectional (Kequ = 37) so that other steps limit the catalytic rate. At saturating substrate concentrations these steps include release of NADP and H4folate from E.NADP.H4folate and release of products from the two abortive complexes E.NADPH.H4folate (koff = 225 s-1) and E.NADP.H4folate (koff = 4.6 s-1). Since NADP dissociates slowly from E.NADP.H2folate nearly 90% of the enzyme accumulates as this complex at steady state. Nonetheless, the catalytic rate is maintained at 12 s-1 by rapid flux of a small portion of the enzyme through an alternate branch. At physiological concentrations of substrates and products the steady-state rate is limited primarily by the rate of H2folate binding to E.NADPH so that the enzyme is extremely efficient.

A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-state kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6----Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the kcat for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the Km values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by alpha-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

Oligonucleotide-directed, site-specific mutagenesis was used to convert phenylalanine-31 of human recombinant dihydrofolate reductase (DHFR) to leucine. This substitution was of interest in view of earlier chemical modification studies (Kumar et al., 1981) and structural studies based on X-ray crystallographic data (Matthews et al., 1985a,b) which had implicated the corresponding residue in chicken liver DHFR, Tyr-31, in the binding of dihydrofolate. Furthermore, this particular substitution allowed testing of the significance of protein sequence differences between mammalian and bacterial reductases at this position with regard to the species selectivity of trimethoprim. Both wild-type (WT) and mutant (F31L) enzymes were expressed and purified by using a heterologous expression system previously described (Prendergast et al., 1988). Values of the inhibition constants (Ki values) for trimethoprim were 1.00 and 1.08 microM for WT and F31L, respectively. Thus, the presence of phenylalanine at position 31 in human dihydrofolate reductase does not contribute to the species selectivity of trimethoprim. The Km values for nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and dihydrofolate were elevated 10.8-fold and 9.4-fold, respectively, for the mutant enzyme, whereas the Vmax increased only 1.8-fold. Equilibrium dissociation constants (KD values) were obtained for the binding of NADPH and dihydrofolate in binary complexes with each enzyme. The KD for NADPH is similar in both WT and F31L, whereas the KD for dihydrofolate is 43-fold lower in F31L. Values for dihydrofolate association rate constants (kon) with enzyme and enzyme-NADPH complexes were measured by stopped-flow techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

The binding site residue Trp-24 is conserved in all vertebrate and bacterial dihydrofolate reductases of known sequence. To determine its effects on enzyme properties, a Trp-24 to Phe-24 mutant (W-24-F) of human dihydrofolate reductase has been constructed by oligodeoxynucleotide site-directed mutagenesis. The W-24-F mutant enzyme appears to have a more open or flexible conformation as compared to the wild-type human dihydrofolate reductase on the basis of results of a number of studies. These studies include competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase, thermal stability, and protease susceptibility studies of both mutant W-24-F and wild-type enzymes. It is concluded that Trp-24 is important for maintaining the structural integrity of the native enzymes. Changes in relative fluorescence quantum yield indicate that Trp-24 is buried and its fluorescence quenched relative to the other two tryptophan residues in the wild-type human reductase. Kinetic studies indicate that kcat values for W-24-F are increased in the pH range of 4.5-8.5 with a 5-fold increase at pH 7.5 as compared to the wild-type enzyme. However, the catalytic efficiency of W-24-F decreases rapidly as the pH is increased from 7.5 to 9.5. The Km values for dihydrofolate are also increased for W-24-F in the pH range of 4.5-9.5 with a 30-fold increase at pH 7.5, while the Km value for NADPH increases only ca. 1.4-fold at pH 7.5 as compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

The transient state kinetics of catalysis for dihydrofolate reductase (DHFR) from several enzyme sources including highly purified recombinant human enzyme (rHDHFR) have been examined. Like DHFR from Escherichia coli, the enzyme from Lactobacillus casei, and isoenzyme 2 from Streptococcus faecium exhibit a slow increase in activity upon addition of substrates to enzyme. No slow hysteresis of this type was detected with recombinant human DHFR (rHDHFR) or DHFR from chicken or bovine liver or L1210 mouse leukemia cells (MDHFR). In contrast, both rHDHFR and MDHFR exhibited a very rapid decrease in activity (t1/2 = 30 and 20 ms, respectively) during a phase that occurred after the first turnover of the enzyme but before establishment of the steady state. This intermediate phase was not observed for the bacterial enzymes or the avian enzyme, nor was it observed with a mutant of rHDHFR in which Phe-31 has been replaced by leucine. For rHDHFR the intermediate phase is not a consequence of product inhibition, substrate depletion, or enzyme instability. It may therefore be concluded that this unusual transient state kinetic behavior results from the existence of two conformers of the enzyme, one of which has a higher turnover number than the other with the equilibrium shifting in favor of the less active conformer during the course of catalysis. The equilibrium is particularly favorable for the less active conformer when NADP is present in the active site of rHDHFR, whereas bound tetrahydrofolate favors the more active conformer. The more active conformer has a 6-fold higher Km for dihydrofolate than does the less active conformer. The existence of these conformers is likely to produce cooperative behavior by rHDHFR in vivo.

Peptides from human dihydrofolate reductase (DHFR) generated by cyanogen bromide cleavage and corresponding to residues 15-52, 53-111, 112-125, and 140-186 (carboxyl terminus) were purified and used to immunize rats. Titration of the immune sera against denatured human DHFR by solid-phase immunoassay showed that peptides 15-52 and 140-186 were relatively highly immunogenic, unlike the native enzyme which is most immunogenic in the sequence 53-111. The antisera were specific for the corresponding peptides used for immunization. Antibodies to peptides 15-52, 53-111, and 140-186 cross-reacted with native human DHFR in solution in competition assays. However, the binding of nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and the inhibitors folate and methotrexate, both in binary and in ternary complexes with the enzyme, caused a striking reduction in binding of antibody. Using a sensitive radioactive assay, it was found that antisera to peptides 15-52 and 140-186, both of which exhibited a high antibody titer, caused significant inhibition of DHFR. Because peptide 140-186 does not include any active-site residues, it is concluded that at least in this case all the antibodies bound to regions outside the active site. Since comparison of the X-ray structures of the chicken liver DHFR holoenzyme with the apoenzyme reveals no changes in secondary structural elements (alpha-helices and beta-sheets), the reduction in antibody binding to DHFR-ligand complexes must not involve epitopes within these structures.(ABSTRACT TRUNCATED AT 250 WORDS)