Metabolic studies with nonnutritive sweeteners cyclooctylsulfamate and 4-methylcyclohexylsulfamate. The nonnutritive sweeteners cyclooctylsulfamate and 4-methylcyclohexylsulfamate were fed separately to female Wistar albino rats, and the urine was examined for the possible metabolites cyclooctylamine, cyclooctanone, cyclooctanol, 4-methylcyclohexylamine, 4-methylcyclohexanone, and cis- and trans-4-methylcyclohexanols. The average percent conversions to cyclooctylamine, cyclooctanone, and cyclooctanol were 0.127, 0.08, and 0.092, respectively. The average percent conversions to 4-methylcyclohexylamine and 4-methylcyclohexanone were 0.007 and 0.0013, respectively. No cis- or trans-4-methylcyclohexanol metabolites were found. With cyclooctylsulfamate, 42% was recovered unchanged from the urine. Cyclooctyl- and cycloheptylsulfamates were metabolized to a greater extent than cyclopentylsulfamate, which, in turn, was metabolized to a greater extent than cyclohexylsulfamate (cyclamate) and 4-methylcyclohexylsulfamate.


GLC determination of purity of Schiff bases bakrine and saddamine. GLC methods were developed for the investigation of impurities in bakrine and saddamine. The method used for bakrine was unsuitable for saddamine since two possible saddamine impurities, benzylamine and salicylaldehyde, reacted very readily in solution to form saddamine, thus giving a false low value for the original concentration of these impurities. The method devised for saddamine involved silylation, which greatly reduced the possibility of saddamine formation from its precursors and also enabled the detection of another possible impurity, salicylic acid. The method described has an obvious application to the determination of other Schiff bases.


Synthesis of 1-phenyl-2-(phenylcarbamoyl)pyrazolidines as potential anticonvulsant agents. Twelve 1-phenyl-2-(phenylcarbamoyl)pyrazolidines were synthesized from 1-arylpyrazolidines and aryl isocyanates. These adducts showed little anticonvulsant activity in the maximal electroshock seizure and pentylenetetrazol seizure assays.


Sensitive high-pressure liquid chromatographic determination of propranolol in plasma. A high-pressure liquid chromatographic method is presented for the determination of propranolol in human plasma. A reversed-phase cyanopropylsilane column was utilized with a liquid phase consisting of 70% acetonitrile and 30% 0.02 M acetate buffer, pH 7.0. A spectrofluorometric detector with an excitation wavelength of 276 nm and an emission filter with a 340-nm cutoff provided a detectable peak for 0.8 ng of propranolol/injection with this system. The reproducibility and precision of the method are shown from the analyses of samples containing 10--150 ng/ml of plasma.


Electron-capture GLC assay of dichlorphenamide. GLC with electron-caputre detection was applied to the assay of the carbonic anhydrase inhibitor dichlorphenamide and demonstrated a sensitivity of 10 ng in 0.5 ml of rabbit serum or whole aqueous humor (congruent to 0.25 ml) from one rabbit eye. After extraction of the drug and internal standard (monochlorphenamide) from the biological fluid, these compounds were converted to their tetramethyl derivatives by a nucleophilic alkylation method. Dichlorphenamide contents of aqueous humor and serum of rabbits treated with this drug are reported.


Comparison of in vitro activity of undecylenic acid and tolnaftate against athlete's foot fungi. Undecylenic acid and tolnaftate were tested in an in vitro test system to evaluate their relative "killing time" efficacy against Trichophyton mentagrophytes, Trichophyton rubrum, and Epidermophyton floccosum. Commercial products containing these active agents were tested similarly. The pure active agents were equivalent in activity. The commercial product containing undecylenic acid appeared to be more effective against the test organisms than did the product containing tolnaftate.


Rapid, sensitive GLC determination of pentobarbital and other barbiturates in serum using nitrogen-specific detector. A GLC method for the analysis of pentobarbital in serum was developed. After extraction from serum, a methyl derivative was prepared and quantitated by nitrogen-specific detection. The method has a senstivity of 0.08 microgram/ml for pentobarbital with only 0.1 ml of serum. Secobarbital was the internal standard. Derivatives of other barbiturates also were prepared.


Separation, identification, and quantitation of anthralin and its decomposition products. Anthralin and its decomposition products were separated by both column chromatographic and TLC techniques. Two decomposition products were characterized by TLC, melting-point data, and UV and IR spectroscopy. Pure anthralin and its decomposition products also were determined quantitatively.


Semantic disorders of auditory language comprehension in right brain-damaged patients. A test of auditory language comprehension was given to 110 right brain-damaged patients and to 94 normal controls in order to check if patients with lesions of the right (nondominant) hemisphere make a significantly higher number of semantic errors than normals. Confirmation of the hypothesis was obtained, but the relationship between semantic errors and lesion of the right hemisphere did not seem a simple and direct one. In fact, most of the lexical-semantic errors were due to associated variables (such as unilateral spatial agnosia and general mental deterioration) and not to the lesion of the right hemisphere per se. These data do not suggest a specific semantic capacity of the nondominant hemisphere but rather stress the fragility of the lexical-semantic organization at the cortical level.


Fertilizing capacity and ultrastructure of fowl and turkey spermatozoa before and after freezing. The fertilizing capacity, motility and ultrastructure of fowl and turkey spermatozoa were examined at various stages of the freezing process. For both species, fertility and motility were depressed after equilibration with dimethyl-sulphoxide at 5 degrees C. After freezing, motility was maintained at 55% for fowl spermatozoa and 40% for turkey spermatozoa; however, fertility was 55% for the fowl and 0% for the turkey. Qualitatively, the damage to the spermatozoa of both species was nearly identical, as revealed by scanning and transmission electron microscopy. The plasmalemma was the primary site of damage. 'Bent' spermatozoa, coiled tails and swollen mitochondria were also present. Damage to the acrosome was only observed in spermatozoa which had been frozen to -180 degrees or -196 degrees C. These changes were attributed to adverse osmotic conditions. Binding of cationic ferritin to the plasmalemma of spermatozoa from both species remained unaltered.


Substrate specificity of the lactate dehydrogenase isoenzyme C4 from human spermatozoa and a possible selective assay. The activity of lactate dehydrogenase (EC 1.1.1.27) in normal human sperm lysates and in human heart and liver homogenates was determined by using a variety of 2-oxoacids as substrates. Sperm preparations were active with pyruvate, 2-oxobutanoate, 2-oxopentanoate and 2-oxohexanoate, while heart and liver extracts utilized only pyruvate and 2-oxobutanoate. Selective staining after gel electrophoresis indicated that the fraction corresponding to lactate dehydrogenase C4, the sperm-specific isoenzyme, was responsible for the utilization of substrates with a linear chain of 3 to 6 carbon atoms. The use of 5 mM 2-oxohexanoate allowed the selective determination of isoenzyme C4 in preparations containing different lactate dehydrogenase molecular forms.


Correlation of lactate dehydrogenase isoenzyme C4 activity with the count and motility of human spermatozoa. The activity of lactate dehydrogenase isoenzyme C4 was determined on 90 human semen samples. The correlation between the isoenzyme activity and sperm count and motility was good (r = 0.74 for values of U/ml semen against sperm count).


Varying effects of an IUD on decidualization in mice. Chronically implanted IUDs consisting of silk suture threads induced decidualization in regions of the uterus remote from the suture site in ovariectomized mice treated with a regimen of progesterone and oestrogen which sensitizes the uterus to a decidual stimulus. In these conditions the IUDs did not inhibit decidualization induced by instilled oil, although they did so in pregnant animals of the same strain. Varying the dose of progesterone and oestrogen did not produce conditions in which IUD's inhibited oil-induced decidualization in ovariectomized mice and progesterone treatment did not prevent IUDs inhibiting decidualization in pregnant animals. However, when ovariectomized mice, sensitized as before, were primed repeatedly with oestrogen to simulate continuing oestrous cycles after IUD insertion, the IUD's inhibited oil-induced decidualization. This involved the premature loss of instilled oil from the uterine lumen and was associated with heavy infiltration of leucocytes into the luminal epithelium. Numbers of leucocytes free in the uterine lumen did not appear to be critical. It appears that contact between the oil and the luminal epithelial surface must be sustained for some length of time to induce a decidual reaction; brief contact is not sufficient to trigger the response.


Early cellular changes and circular muscle contraction associated with the induction of decidualization by intrauterine oil in mice. Intrauterine instillations of oil or saline distended the uterus in ovariectomized mice treated with progesterone + oestrogen to sensitize the uterus to a decidualizing stimulus. Saline does not induce decidualization, and therefore uterine distension per se is not the trigger to decidual induction. Oil induces decidualization, but does not involve gross damage to the epithelium, penetration of oil into the stroma or release of epithelial lipid into the stroma. Instillation (oil, saline or sham) induced a contraction of the circular muscles along the length of the uterus which closed the uterine lumen, expelled most of the oil and located the remainder primarily in the antimesometrial cleft of the lumen. Progesterone inhibited longitudinal muscle contraction and facilitated circular muscle contraction. These effects are discussed in relation to the spacing and implantation of blastocysts.


Symplasma formation and decidualization in the pseudopregnant rabbit after intraluminal instillation of tricaprylin. A pure neutral triglyceride, tricaprylin, was injected (Day 0) into isolated segments of the uterus of rabbits in which pseudopregnancy had been induced by i.v. injection of hCG 6 days before. The animals were killed in groups of 3 on Days 1, 2, 3, 4 and 6. Saline, simple ligation of the uterus or uterine trauma were used as control treatments in other uterine segments in the same animals. Extensive symplasma formation in the uterine epithelium of the tricaprylin-treated segments was observed on Days 2 and 3; this had degenerated by Day 4 and was absent by Day 6. Decidualization of the stroma began on Days 3 and 4 and was absent by Day 6. The symplasma formed earlier on the antimesometrial side than it did laterally or mesometrially. Decidualization was maximal mesometrially, intermediate laterally, and absent antimesometrially. Ligation with saline and trauma, but not ligation alone, produced symplasma formation. Decidualization occurred after trauma but not after saline injection or ligation alone.


Thromboxane B-2 in the plasma and amniotic fluid of late pregnant and periparturient sheep. The concentration of thromboxane (TX) B-2 was similar in plasma from all sources but was significantly greater (P less than 0.01) in amniotic fluid. Fetal hypophysectomy was without effect on maternal or fetal levels of TXB-2 (P greater than 0.1). Neither normal parturition at term nor prematurely induced delivery was associated with any significant trend in TXB-2 levels. During late pregnancy (105--145 days of gestation) the concentrations of TXB-2 and 13, 14-dihydro-15-keto-prostaglandin F (PGFM) in maternal and fetal plasma were significantly correlated (P less than 0.001). There was, however, no correlation between TXB-2 and PGFM levels in samples taken during the 36 h before delivery. These data suggest that thromboxanes play little part in the mechanism of parturition in sheep.


The estimation of sperm motility in semen, on a membrane slide, by measuring the area change frequency with an image analysing computer. A simple slide is described in which the base is a permeable membrane so that a suspension of spermatozoa (or other cells) may be examined under controlled conditions with a microscope. An objective method of assessing the motility of spermatozoa in undiluted or diluted semen from a wide variety of species including cattle, horse, pig, rabbit, rat and sheep is described. It is shown to be well correlated with other methods of assessing motility and also with the non-return rate obtained with frozen cattle semen.


Measurement of the motility of rat spermatozoa collected by micropuncture from the testis and from different regions along the epididymis. Spermatozoa from the testis and various regions along the epididymis of the rat were collected by micropuncture and their motility after dilution was estimated over a 15-min period by using a Quantimet image analyser. The motility of sermatozoa from the rete testis and seminiferous tubules was too low to be measured. The estimate of motility of spermatozoa from the proximal caput epididymidis was much lower than that of spermatozoa from the other regions. Spermatozoa from the distal part of the caput showed sustained motility for 15 min, whereas those from the caudal region and ductus deferens, although active initially, became less active during this period.


Hormonal and physical changes associated with bovine conceptus development. Conceptus (placental membranes, fetal fluids and fetus) development was characterized between Days 27 and 111 of gestation. Progestagens, oestrone, oestradiol, oestrone sulphate and prostaglandins (PG) F were measured in maternal plasma and allantoic and amniotic fluids. Protein concentrations are described for fetal fluids. The early increase in placental membrane weight from 1.12 g (27 days) to 58.45 g (50 days) was associated with oestrogen production presumably of conceptus origin. Oestrogens increased significantly in allantoic and amniotic fluids throughout the period studied with oestrone being the primary free oestrogen, rising from 2 pg/ml (Day 33) to 144 ng/ml by 111 days in allantoic fluid. Changes in plasma oestrogens of the maternal circulation were not detected until after Day 70 at which time oestrone concentration was greater than that of oestradiol. Fetal fluid concentrations of progestagens, oestrone sulphate and PGF were not related to maternal plasma levels and a sequestration of these hormones by the allantois is postulated.


Regeneration of atretic sheep ovarian follicles in vitro. Large (4--6 mm diam.) and small (2--3 mm) atretic follicles were removed from sheep ovaries during the luteal phase of the cycle and maintained in organ culture without hormonal supplementation for up to 5 days. The structure, cell dynamics and steroid-producing capacity of the follicles were compared with those of non-atretic follicles of similar size. The granulosa layer of the atretic follicles invariably regenerated in culture, increasing in thickness more than 2- and 4-fold in large and small follicles respectively. This could not be accounted for by cell division which remained low throughout the culture period. In contrast, non-atretic follicles showed high mitotic activity during the first 24 h in culture: this was not associated with an increase in granulosa thickness in large follicles although there was a 4-fold increase in small ones. An increase in internuclear spacing, a measure of cell size plus intercellular space, partly accounted for the increase in granulosa thickness in atretic follicles. Even when granulosa cells remained in close apposition there was an almost total absence of gap junctions, a prominent feature in the granulosa of non-atretic follicles both in vivo and in vitro. Pyknotic nuclei and atretic bodies rapidly disappeared from the regenerating granulosa layer. The theca interna was restored in culture to a state ultrastructurally closely resembling that of non-atretic follicles in vivo. Total steroid secretion (oestradiol-17beta, testosterone plus progesterone) into the culture medium (pmol.mg tissue-1.24 h-1) was the same for atretic and non-atretic follicles of comparable size. There was, however, a marked difference in the type of steroid produced, largely related to a loss of aromatizing capacity in atretic follicles. The predominant steroid secreted by large non-atretic follicles was oestrogen, with slightly smaller amounts of testosterone, whereas the principal steroid secreted by large atretic follicles was progesterone. In small non-atretic and atretic follicles, the predominant steroid was testosterone, but the non-atretic follicles also secreted appreciable amounts of oestrogen. Addition of FSH to the culture medium did not restore aromatizing capacity to the atretic follicles.


Total follicular populations in ewes of high and low ovulation rates. The total ovarian follicular populations were studied in two breeds of ewes which differed greatly in their ovulation rates. Thus 8 Romanov (mean ovulation rate 3.1) and 12 Ile-de-France ewes (mean ovulation rate 1.4) were ovariectomized at oestrus during the breeding season. Each right ovary and 3 left ovaries were sectioned at 7 micron and examined microscopically. The number of small follicles, i.e. with 2 or less layers of granulosa cells, was estimated by a tested sampling procedure whilst all larger follicles were measured and arranged into classes. There were half as many small follicles but 1.5--2 times more large follicles in the ovaries of the Romanov ewes compared to those of the Ile-de-France ewes. The number of atretic follicles was approximately the same in both breeds and does not explain the difference observed in ovulation rate. It is concluded that the higher ovulation rate in the Romanov ewe is due to the greater number of large follicles available to be stimulated for ovulation.


A study of endometrial RNA polymerase activity in infertile women. Endometrial RNA polymerase activity, which is sensitive to oestrogens and progesterone, has been measured in normal and infertile patients. One-third of the infertile patients studied had abnormal values. An analysis of the relationship of abnormal RNA polymerase activity to clinical abnormalities and to pregnancies occurring without treatment suggests that this assay may detect a biochemical fault in the endometrium which is related to the failure to conceive.


Effect of ovulation and sperm motility on the migration of rabbit spermatozoa to the site of fertilization. Few spermatozoa were present in the ampullae of females 12 h after intravaginal artificial insemination (AI) when there was no ovulation-inducing stimulus. When ovulation was induced, sperm distributions in the female tract 12 h after AI did not differ from those observed 12 h after natural mating. The number of spermatozoa in the oviductal isthmus was similar in all 3 groups as was the percentage of isthmic spermatozoa exhibiting 'activated' motility. When fertile mating was delayed for 8 or 12 h after coitus with a vasectomized male (i.e. 2 h before or after ovulation), spermatozoa were not present in the ampulla 4 h later. The numbers of spermatozoa recovered from the cranial isthmus after delayed matings and 12 h after natural matings did not differ, but after delayed matings the motility of isthmic spermatozoa was non-progressive or poorly progressive and none exhibited 'activated' motility. Flagellar activity of isthmic spermatozoa recovered 4 h after delayed matings and after natural matings was similarly depressed. These observations indicate that sperm ascent to the tubal ampulla in the sustained phase of transport, though enhanced by ovulation, must also depend on changes in flagellar activity and a specific pattern of motility, both of which appear only after spermatozoa have resided for more than 4 h in the female tract.


Oviposition of fertile eggs and postponement of subsequent egg laying in ring doves (Streptopelia risoria). Within the 45 days after an oviposition, fewer female doves which had laid fertile eggs laid a subsequent clutch than did females which had laid infertile eggs. During incubation the fertile eggs were replaced by infertile ones; therefore, this difference was not a result of raising squabs.


Testosterone and 5alpha-dihydrotestosterone production in vitro by rat testicular tissue treated with oestradiol-17beta. Decapsulated adult rat testes were assessed for their capacity to produce testosterone and 5alpha-dihydrotestosterone when incubated in the presence of oestradiol-17beta for 3 h. Concentrations of 10(-6) and 10(-8) M-oestradiol-17beta had no significant effect on the production of these hormones and did not alter the capacity of the testes to respond to 100 i.u. hCG in vitro. It is suggested that oestradiol-17beta does not directly affect acute regulation of testicular steroidogenesis in the adult rat.


Effects of phenylmethylsulphonylfluoride on activities of cholesteryl ester synthesis and hydrolysis in testes of rats, and on serum testosterone and LH levels. PMSF was injected subcutaneously to male rats once a day for 10 days at a dose of 10 mg/day. There was an increase in the concentrations of free and esterified cholesterol in the testes, a decrease in activities of cholesteryl ester synthesis and hydrolysis but no change in activity of cholesterol side-chain cleavage enzyme. Serum testosterone and LH levels were significantly decreased. The only effect of PMSF on the lipids of the testes was a marked elevation of docosapentaenoic (22:5) acid and the cholesteryl esters.


Effects of season on the secretion of LH and testosterone in intact and castrated red deer stags (Cervus elaphus). At 2--4 monthly intervals during the year blood samples were collected every 15 min for 6 h from 2 intact and 3 castrated red deer stags to study the relationship between season and the secretion of LH and testosterone. In the intact stags plasma LH and testosterone concentrations changed during the year; the LH levels were maximal in August during the phase of testicular redevelopment, while the testosterone levels were maximal from September to November coinciding with the time of peak testicular activity and the mating season. The castrated stags had higher plasma levels of LH than the intact stags at all times of the year, and there was no clear seasonal cycle in LH levels in these animals.


Short-term variations in plasma LH and testosterone in bull calves from birth to 1 year of age. LH and testosterone levels in bull calves were studied in the plasma samples collected sequentially at 15-min intervals every month during the first year of life. An episodic pattern of LH release occurred after birth and the frequency and magnitude of the LH peaks increased up to 4 months of age and decreased thereafter. A testicular response was not observed before this age. It is suggested that this episodic LH activity is responsible for the testicular development which then initiates puberty.


Ovulation rate and oocyte numbers in ewes after prolonged exposure to oestrogenic pasture. Ewes which had been exposed to oestrogenic clover for 3 years showed an elevated ovulation rate but no difference in numbers of primordial follicles compared with normal ewes.


Analysis of mouse uterine proteins at pro-oestrus, during early pregnancy and after administration of exogenous steroids. Uterine secretions were analysed after labelling with L-14,5-3H]leucine. During pro-oestrus major protein peaks were seen with approximate mol. wt 2.0 X 10(4), 2.7 X 10(4), 4.0 X 10(4), 5.7 X 10(4), 7.8 X 10(4) and 13.0 X 10(4). Qualitative changes were observed during the first 4 days of pregnancy. On day 4 p.c. secretions revealed two prominent peaks with mol. wt 6.7 X 10(4) and 12.5 X 10(4), four peaks between 2.0 and 4.7 X 10(4) and one at 8.5 X 10(4) and one at 20.0 X 10(4). Profiles similar to those on mimic Day 4 p.c., and implantation, were induced in ovariectomized females by administration of exogenous progesterone and oestradiol, whereas the proteins detected after administration of exogenous oestradiol alone were essentially similar to those found at pro-oestrus. Ovariectomized mice treated with the steroid sequence to mimic Day 4 p.c. profiles but not given the final injection of oestradiol showed no detectable labelled proteins. Injection of progesterone or oestradiol alone, in the final sequence of injections given to ovariectomized pregnant females treated with progesterone, did not induce blastocyst implantation. These studies indicate a synergistic relationship between progesterone and oestrogen in the secretion of uterine macromolecules.


Fitz-Hugh and Curtis syndrome.

Fitz-Hugh and Curtis syndrome. A review of 124 patients treated for pelvic inflammatory disease revealed 15 cases of presumptive Fitz-Hugh and Curtis syndrome. During the same 12-month period three patients mistakenly thought to have gall bladder disease came to laparotomy, only to be found to have Fitz-Hugh and Curtis syndrome. The subtle presentation of the syndrome is discussed.


Synthesis and evaluation for diuretic activity of 1-substituted 6-chloro-5-sulfamylindolines. The synthesis of a series of 1-substituted 6-chloro-5-sulfamylindolines is described. In the Lipschitz test for diuretic activity, two of the compounds showed significant excretion of urine and sodium and were approximately equivalent in potency to chlorothiazide but with a later onset of activity.


Phosphonate analogue of 2'-deoxy-5-fluorouridylic acid. The phosphonate analogue (6) of 2'-deoxy-5-fluorouridylic acid has been prepared via a Pfitzner--Moffatt oxidation and Witting reaction. This compound was found to inhibit thymidylate synthetase from three sources and to be cytotoxic to H.Ep.-2 cells in culture.


Correlation of carbonic anhydrase inhibitory activities of benzenesulfonamides with the data obtained by use of nitrogen-14 nuclear quadrupole resonance. Nitrogen-14 nuclear quadrupole resonance (NQR) spectra of several benzenesulfonamides in their solid state are reported and analyzed in the framework of the Townes and Dailey theory. Satisfactory correlations between the (sigma NH--sigma NS) electron densities at the sulfamyl nitrogen and the in vitro carbonic anhydrase inhibitory activities of the sulfonamides have been found. The correlations are in accord with the results of other studies that show the carbonic anhydrase inhibitory activities to be largely influenced by the electronic property of the sulfamyl group.


11-Oxo-11H-pyrido[2,1-b]quinazoline-8-carboxylic acid, an orally active antiallergy agent. A new series of 11-oxo-11H-pyrido[2,1-b]quinazolinecarboxylic acids and related analogues has been synthesized and evaluated as potential antiallergy agents. In the rat PCA test, 11-oxo-11H-pyrido[2,1--b]quinazoline-8-carboxylic acid is orally active and more potent than cromolyn sodium or doxantrazole intravenously.


Actions of two dopamine derivatives at adreno- and cholinoceptors. Salsolinol (1)and 6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline hydrobromide (2) were synthesized and their effects at adreno- and cholinoceptors investigated both in vivo and in vitro. Both 1 and 2 produced agonist effects at cholinoceptors and alpha- and beta-adrenoceptors. Neuromuscular blocking actions were evident in vitro. Compound 2 exhibited anticholinesterase properties both in vivo and in vitro. These results indicate that dopamine derivatives of this type exhibit not only sympathomimetic activity but also complex actions at cholinoceptors.


Apparent bioisosteric replacement of--S--by NCN: synthesis of N--cyano-2-aza-A-nor-5alpha-androstan-17beta-ol acetate, an aza steroid androgen. The synthesis of N-cyano-2-aza-A-nor-5alpha-androstan-17beta-ol acetate is described. Cyclization of 1,4-dibromo-1,4-seco-2,3bisnor-5alpha-androstan-17beta-ol acetate with benzylamine in the presence of potassium iodide gives the N-benzyl-2-aza-A-nor steroid. Debenzylation with cyanogen bromide (Von Braun reaction) affords the N-cyano-2-aza-A-nor steroid, which has androgenic activity slightly weaker than that of the corresponding thia compound. The results indicate that NCN may be substituted for--S--as well as for =S. This compound is the first hormonally active steroid containing nitrogen as a heteroatom in the perhydrocyclopentanophenanthrene nucleus.


Molecular orbital study on the reaction mechanism of irreversible enzyme inhibitors. By means of the molecular orbital method, the reaction mechanism of the specific and irreversible enzyme inhibitors, such as cycloserine, L-2-amino-4-methoxy-trans-3-butenoic acid (AMB), and vinylglycine (2-amino-3-butenoic acid), was studied. Firstly, it was attempted to know which pathway is probable between the transamination process and the isomerization one. By comparing the energy increments for these two reactions, the transamination reaction was predicted to be energetically favorable, supporting the proposition of Rando. Upon complexing with the coenzyme-pyridoxal moiety of alanine racemase or aminotransferase, the reactivity of the inhibitors toward the nucleophile was found to be considerably increased due to the lowering of the lowest unoccupied molecular orbital (LUMO), and this was considered to be the reason why the inhibitors become bound with the enzyme irreversibly. The LUMO of aspartate, substrate of aspartate aminotransferase, is higher than those of the inhibitors in the free state, as well as in the pyridoxal-linked state. This difference in the energy of the molecular orbital between substrate and inhibitors was considered to be correlated with the difference in the complex-forming properties of these compounds toward the nucleophile in the enzyme.


Structure-activity relationship in a new series of atropine analogues. 1. N,N'-Disubstituted 6,7-diazabicyclo[3.2.2]nonane derivatives. The synthesis of a new series of N,N'-disubstituted 6,7-diazabicyclo[3.2.2]nonane derivatives is described. The antimuscarinic potency of these drugs was evaluated in the guinea pig ileum and compared to that of atropine sulfate. All the drugs tested competitively inhibited the acetylcholine-induced contractions. Kd values were calculated and, in several cases, compared to those obtained by direct binding to the muscarinic receptor from mouse brain. The order of potencies followed that which is known for various tropine and pseudotropine esters; that is, the 3alpha configuration is more potent than the 3beta configuration, and the quaternary analogues are more potent than the tertiary ones. The antimuscarinic activity of the drugs is dicussed in terms of their acetylcholine-like molecular arrangement that gives rise to a characteristic interaction pharmacophore.


Antileukemic activity of substituted ureidothiazoles, ureidothiadiazoles, and related compounds. A number of ureidothiazole and ureidothiadiazole derivatives related to ethyl 4-[[(2-thiazolylamino)carbonyl]-amino]benzoate were prepared and evaluated against the leukemia P-388 tumor system in mice. Preliminary structure-activity relationship study revealed that, among other considerations, active compounds of this series contain either an "isothioureido" [N-C(S-)=N-] or an "isothiosemicarbazono" [N-C(S-)=N-N=] structural unit.


Ergot alkaloids. Synthesis of nitrosourea derivatives of ergolines as potential anticancer agents. Nitrosourea derivatives of ergolines have been synthesized for the purpose of obtaining agents with both prolactin-and tumor-inhibitory activity. Two derivatives of 8-amino-6-methylergoline (3), 8-[3-(2-chloroethyl)-3-nitrosoureido]-1-nitroso-6-methylergoline (5c) and 8-[3-2-chloroethyl)-3-nitrosoureido]-6-methylergoline (5a), have been prepared. In addition, nitroso (7) and chloroethylcarbamyl (8) derivatives of elymoclavine (6) are reported. Compounds 5a and 5c have activity against L1210 leukemia in mice but only moderate prolactin-inhibiting activity. The chloroethylcarbamyl derivative 8 of elymoclavine is a potent prolacting inhibitor.


5-Iminodaunorubicin. Reduced cardiotoxic properties in an antitumor anthracycline. Treatment of daunorubicin with methanolic ammonia affords 5-iminodaunorubicin, the first quinone-modified analogue of either daunorubicin or adriamycin. This product retains antileukemic activity in mice, is less cardiotoxic by electrocardiographic measurements in rats, and is nonmutagenic in Salmonella typhimurium (Ames test).


Synthesis of daunorubicin analogues with novel 9-acyl substituents. Synthetic approaches to anthracyclines bearing novel 9-acyl substituents were investigated. Reaction of the lithium enolate of N-(trifluoroacetyl)daunorubicin (9) with methyl iodide in tetrahydrofuran afforded only the 9-propionyl derivative 10 in high yield. Reaction of 10 under identical conditions cleanly afforded the 9-isobutyryl derivative 11. Extension of this procedure to other alkylating agents (ethyl iodide, benzyl bromide, and heptyl iodide) required hexamethylphosphoramide as cosolvent and afforded mixtures of mono- and dialkylated products as well as recovered 9. The amino group was deblocked with NaOH in aqueous tetrahydrofuran, except in the case of the dibenzyl derivative 13 which was inert under these conditions. The 9-formyl analogue 23 was prepared via NaIO4 cleavage of 13-dihydroadriamycin (21). Antitumor evaluation against P388 leukemia in mice showed 23 to have activity comparable to the parent compounds, while the C-alkylated analogues were less active.


Syntheses and antiinflammatory actions of 4,5,6,7-tetrahydroindazole-5-carboxylic acids. A novel series of 1-aryl-4,5,6,7-tetrahydro-1H-indazole-5-carboxylic acids and 2-aryl-4,5,6,7-tetrahydro-2H-indazole-5-carboxylic acids were synthesized via condensation between a phenylhydrazine and a 2-(hydroxymethylene)cyclohexanone-4-carboxylate, and the antiinflammatory activity was determined. In the carrageenan edema test, 1-aryl-4,5,6,7-tetrahydro-1H-indazole-5-carboxylic acids exhibited fairly high antiinflammatory activity. However, the 2-aryl isomers were far less active than the former. The most active compound of the series was 1-phenyl-4,5,6,7-tetrahydro-1H-indazole-5-carboxylic acid, which had an ED50 value of 3.5 mg/kg.


Synthesis and evaluation of the antiovulatory activity of a variety of melatonin analogues. A series of melatonin analogues was synthesized and examined for ovulation-blocking activity. Deviation from the 5-methoxy group or substitution of the 1 position prevented activity. Activity was not particularly sensitive to minor variations in the N-acyl group nor was it significantly altered by methylation of position 2 or the alpha-methylene; however, a pronounced enhancement resulted from halogenation of the 6 position.


[1-Penicillamine,2-leucine]oxytocin. Synthesis and pharmacological and conformational studies of a potent peptide hormone inhibitor. [1-Penicillamine,2-leucine]oxytocin was synthesized by the solid-phase method of peptide synthesis and purified by partition chromatography on Sephadex G-25, followed by gel filtration. The peptide was found to be a very potent competitive inhibitor of oxytocin in the oxytocic assay with a pA2 of 7.14 and an inhibitor of oxytocin in the milk-ejecting assay. The compound showed no agonist activity in either of these assays, and its inhibitory activity at the uterus was of prolonged duration. The 13C nuclear magnetic resonance spectral properties and the 13C T1 (spin-lattice) relaxation times of [Pen1,Leu2]oxytocin were determined, and the results were compared with previous studies of [Pen1]oxytocin, a related competitive inhibitor, and oxytocin, the native hormone agonist. These studies indicated that the hormone inhibitors [Pen1,Leu2]oxytocin and [Pen1]oxytocin have similar conformational and dynamic properties which are different than those of the agonist, oxytocin.


2-Indanpropionic acids: structural leads for prostaglandin F2alpha antagonist development. A rationale is presented for the development of prostaglandin F2alpha receptor antagonists. The target analogue, 5,6-(dibenzyloxy)-1-oxo-2-propyl-2-indanpropionic acid (3), was shown to have selective activity for antagonism of PGF2alpha when compared to the antagonism of acetylcholine and KCl on the mouse ileum, whereas other 2-indanpropionic acids (1, 2, 4), not substituted with benzyl functions, were considerably less active and nonselective. The results suggest that 3 may serve as a lead compound for further drug development.


Relationship of nonspecific antiarrhythmic and negative inotropic activity with physicochemical parameters of propranolol analogues. In an attempt to separate the nonspecific antiarrhythmic activity of propranolol from its negative inotropic effects, analogues containing hydrophilic and lipophilic substituents on the nitrogen and on the naphthyl ring were prepared and tested in an isolated tissue preparation. Though it had been predicted that analogues containing a very hydrophilic group on the nitrogen would have the highest antiarrhythmic/negative inotropic effect ratio, it was found that both effects increased identically when the lipophilicity of either the nitrogen or ring substituent was increased.


Discriminant--analytical investigation on the structural dependence of hyperglycemic and hypoglycemic activity in a series of substituted o-toluenesulfonylthioureas and o-toluenesulfonylureas. The influence of a series of substituted o-toluenesulfonylthioureas and o-toluenesulfonylureas on the level of blood sugar was investigated in rats. According to the observed response the compounds were divided into three classes corresponding to hypoglycemic, hyperglycemic, and no activity. The distribution of the compounds over these classes can be described by discriminant functions using substituent constants, RM values, and indicator variables. Most important for the separation of classes are hydrophobic and/or steric properties as well as the presence or absence of the thiomide group. The results indicate that two different mechanisms of action with opposite effect overlap in the case of the series studied.


Studies on biologically active halogenated compounds. 1. Synthesis and central nervous system depressant activity of 2-(fluoromethyl)-3-aryl-4(3H)-quinazolinone derivatives. Some 2-(fluoromethyl) analogues of 2-methyl-3-aryl-4-(3H)-quinazolinones have been synthesized and screened for CNS activities. It was shown that the 2-(fluoromethyl) analogues possess in general more potent CNS depressant activities and less toxicities than their parent compounds. Of particular interest were the 2-(fluoromethyl) analogues (22, 24, and 31) of methaqualone and 6-aminomethaqualone. Compound 24 was more potent in CNS depressant activity and less toxic than methaqualone. Compound 31 exhbited potent central muscle relaxing activity and markedly reduced toxicity as compared with 6-aminomethaqualone.


Kojic amine--a novel gamma-aminobutyric acid analogue. A series of compounds containing the 3-hydroxy-4H-pyran-4-one nucleus has been synthesized and tested as potential skeletal muscle relaxants. Reduction of 2-(azidomethyl)-5-hydroxy-4H-pyran-4-one (4) with HBr in HOAc--phenol yielded 2-(aminomethyl)-5-hydroxy-4H-pyran-4-one (kojic amine, 3) in 81% yield. Reaction of 2-[(tosyloxy)-methyl]-5-(benzyloxy)-4H-pyran-4-one (5) with NH3 gave a 40% yield of the O-benzyl ether of kojic amine, which was N-acylated with a series of carbobenzyloxy-protected amino acids. Complete deprotection with HBr--HOAc gave the following amino acid amides of kojic amine: glycyl (23), alpha-alanyl (24), beta-alanyl (25), gamma-aminobutyryl (26), and glycylglycyl (27). Among the analogues of kojic amine prepared was a series of one-carbon homologues: 2-[(methylamino)methyl]-5-hydroxy-4H-pyran-4-one (7a), 2-(1-aminoethyl)-5-hydroxy-4H-pyran-4-one (8), 6-(aminomethyl)-3-hydroxy-2-methyl-4H-pyran-4-one (12), and 2-(2-aminoethyl)-5-hydroxy-4H-pyran-4-one (16). Kojic amine (3) has been found to possess certain of the properties to be expected in a gamma-aminobutyric acid mimetic agent, notably skeletal muscle relaxant activity. In the chronic spinal cat preparation, ED70 values for reduction of flexor spasms of 2.2 and 4.0 mg/kg by iv and po routes of administration, respectively, were observed for kojic amine, which was the most potent of the various hydroxypyrone derivatives investigated.


Structural requirements for progestational activity. Synthesis and properties of rac-8alpha,9beta,10alpha,14beta-progesterone. rac-8alpha,9beta,10alpha,14beta-Progesterone, 1, has been synthesized and subjected to X-ray crystallographic analysis which established that the ring conformations are A, 1beta-sofa; B, chair; C, chair; and D, intermediate between an envelope and a half-chair. This compound is 10% as active as progesterone in the Clauberg assay and has an affinity for the uterine cytosol (rabbit) receptor for progesterone 2% as great as that of progesterone.


Potential antitumor agents. 29. Quantitative structure-activity relationships for the antileukemic bisquaternary ammonium heterocycles. Quantitative relationships between physicochemical drug properties and antileukemic (L1210) efficacy have been examined for a series of bisquaternary ammonium heterocycles employing multiple variable regression analysis. Three measures of biologic response were examined: ILSmax, the percentage increase in mean life span of leukemic animals at the LD10 dose; D40, the drug dose necessary to provide 40% increase in life span; and CI (=LD 10/D40), the chemotherapeutic index. A cross correlation matrix between these three measures and the LD10 values demonstrates ILSmax and CI to be independent of toxicity. D40 is highly inversely correlated with LD10 and positively correlated with ILSmax, suggesting that this parameter measures a composite of both drug selectivity and toxicity. Superior regression equations resulted at all stages employing ILSmax as a measure of antitumor selectivity. Acceptable equations modeling LD10 could not be obtained. There was a parabolic relationship between agent lipophilic-hydrophilic balance, measured as chromatographic Rm values, and ILSmax. To reduce residual variance in the L1210 screening data, not accepted by this parabolic equation, measures of agent-DNA interaction were investigated as possible indices of site fit. Relative levels of drug-DNA interaction were obtained by spectrofluorimetric quantitation of drug displacement of DNA-bound ethidium. Addition to regression equations of agent C50 values for calf thymus DNA, those micromolar drug concentrations necessary to displace 50% of the ethidium bound to that DNA, provided a significant reduction in the screening data variance. C50 values for drug interactions with poly[d(A-T)] and poly[d(G-C)] were also investigated as possible indicators of drug selectivity towards different DNA sites. Marked differences were observed in the C50 values for the two synthetic nucleic acids, with those for calf thymus DNA and poly[d(G-C)] proving highly covariant. A regression equation containing a positive term in C50[poly[d(G-C)]] and a negative term in C50-[poly[d(A-T)]] provided the greatest acceptance of the variation in the biologic data. The term in C50[poly[d(A-T)]] is the single most important equation term, alone accounting for 35% of the variance in the data. This best equation provides both an adequate summary of the L1210 screening data for the 174 active compounds considered and a physical explanation for the antitumor selectivity for these agents. The major factor influencing antitumor selectivity in these drugs is their ability to distinguish alternating adenine-thymine sequences in DNA.


Optically pure (+)-nicotine from (+/-)-nicotine and biological comparisons with (-)-nicotine. Optically pure (+)-nicotine has been obtained from (+/-)-nicotine using a combination of d-tartaric acid and di-p-toluoyl-l-tartaric acid. As the di-d-tartrate salt, (+)-nicotine is less potent than (-)-nicotine di-l-tartrate in producing lethality in mice, on blood pressure in anesthetized rats, and in the isolated guinea-pig ileum, indicating substantial stereospecificity for nicotine receptors. Potency ratios are 0.14, 0.06, and 0.019, respectively.


Synthesis of pyridinium analogues of acetylcholine and their interactions with intestinal muscarinic receptors. N-(beta-Acetoxyethyl)pyridinium salts were synthesized and tested for muscarinic receptor interactions by the guinea pig ileum assay. Agonist activity indicates that receptor binding is substantially retained when the ammonium group of acetylcholine is formally replaced by a pyridinium ring. Introduction of alkyl groups into the ring yields antagonists. The 4-tert-butylpyridinium derivative is proved to have an activity superior to that of the 4-methylpyridinium salt. Competitive antagonism is favored by the more hydrophobic property of the tert-butyl group. A nonpolar area is suggested to be situated in the direct vicinity of the anionic binding sites of muscarinic receptors. The interaction of hydrophobic substituents with this area determines the antimuscarinic properties of pyridinium salts.


Studies on 3-substituted 1,2-benzisoxazole derivatives. 6. Syntheses of 3-(sulfamoylmethyl)-1,2-benzisoxazole derivatives and their anticonvulsant activities. Several 3-(sulfamoylmethyl)-1,2-benzisoxazole derivatives were synthesized from 3-(bromomethyl)-1,2-benzisoxazole by the reaction with sodium bisulfite followed by chlorination and amination. Some of them displayed marked anticonvulsant activity in mice. The introduction of a halogen atom to the 5 position of the benzisoxazole ring caused increased activity and neurotoxicity; the substitution of a sulfamoyl group caused decreased activity. The activity of monoalkylated compounds might be the result of biotransformation. Among these compounds, 3-(sulfamoylmethyl)-1,2-benzisoxazole (1a) was thought to be the most promising as an anticonvulsant from the ratio of NTD50 and ED50.


Azetidine derivatives of tricyclic antidepressant agents. Tricyclic derivatives of azetidine were synthesized and screened for their potential antidepressant activity. The active series had the tricyclic ring attached to position 1 and a basic group in position 3 of the azetidine. The most interesting compounds were comparable to the reference standards for reserpine antagonism in mice, the most active being the dextrorotatory methylamino derivative 84. The pharmacological profile classifies it as a CNS stimulant devoid of peripheral anticholinergic activity.


Electrochemical oxidation of hydroxylated phenothiazine and imipramine derivatives. The electrochemical oxidations of several hydroxylated derivatives of promazine, chlorpromazine, imipramine, and 3-chloroimipramine are examined and compared. Oxidation of the monohydroxyphenothiazine derivatives leads to both dihydroxy species and substituted benzoquinones, while oxidation of hydroxylated imipramines leads to only the corresponding benzoquinones. The oxidation potentials of 17 tricyclic psychoactive drugs and metabolites are tabulated and compared. The potential importance of these results to drug activity and side effects is discussed.


Phototoxicity of chlorpromazine.

Phototoxicity of chlorpromazine. The constitution of chlorpromazine has been studied in the context of its phototoxicity. Electron transfer from the side chain to the aromatic nucleus of the drug contributes to its instability to light. Even without the side chain, however, chlorophenothiazines appear to be very photolabile, so that it is unlikely that nonphototoxic analogues of chlorpromazine can be prepared merely by altering the constitution of the side chain.


Aminotetralins as narcotic antagonists. Synthesis and opiate-related activity of 1-phenyl-2-aminotetralin derivatives. The synthesis and the opiate agonist and antagonist activities of three derivatives of cis-2-[methyl(cyclopropanemethyl)amino]-1-phenyltetralin are reported. The compounds were obtained by synthetic modification from 2-amino-1-tetralone. The 1-propionoxy derivative 4c shows analgetic activity (ED50 = 17.8 mg/kg) one-half that of codeine, and the 1-methoxy derivative 4b has weak antagonist activity (AD50 = 33.5 mg/kg). The compounds showed no other significant opiate-related activity.


Bicyclic pyrazolines, potential central nervous system depressants and antiinflammatory agents. The synthesis and CNS activity of a series of 34 substituted bicyclic pyrazolines are described. Ten of these compounds were also screened for antiinflammatory activity. One of the compounds (15) exhibited significant antiinflammatory activity in the carrageenan-induced edema test.


Coenzyme--substrate adducts as inhibitors of mouse liver 3,4-dihydroxyphenylalanine decarboxylase. N-(5'-Phosphopyridoxyl) derivatives of several aromatic amino acids have been prepared by conventional methods and tested as inhibitors of mouse liver L-3,4-dihydroxyphenylalanine (Dopa) decarboxylase (EC 4.1.1.26; L-aromatic-amino-acid decarboxylase). The L-tyrosine, L-phenylalanine, and DL-2-hydroxyphenylalanine derivatives were effective inhibitors at concentrations of 10(-5) M. Because of the spontaneous formation of a tetrahydroisoquinoline cyclic condensation product with pyridoxal phosphate (Pictet--Spengler reaction), the Dopa derivative could not be prepared by the usual procedures. The synthesis of the desired N-(5'-phosphopyridoxyl)-Dopa was accomplished using selective blocking--deblocking methods; its properties are described. This proved to be the most effective inhibitor of those tested. Neither the tetrahydroisoquinoline of L-Dopa and pyridoxal phosphate nor the N-(5'-deoxypyridoxyl)-Dopa was an effective inhibitor of Dopa decarboxylase. These coenzyme amino acid adducts are suggested to act as stage inhibitors of the enzyme.


Cyclic amidine inhibitors of indolamine N-methyltransferase. Syntheses of a large number of mono- and bicyclic, as well as a few tricyclic, amidine derivatives related to 2,3,4,6,7,8,-hexahydropyrrolo[1,2-a]pyrimidine (DBN) are reported. In vitro potencies for inhibition of the enzyme indolamine N-methyltransferase (INMT) from rabbit and human lung are presented. Four bicyclic amidine derivatives and 11 monocyclic derivatives were found to be equal or superior to DBN in in vitro potencies. With the bicyclic amidines, increasing ring size or introduction of substituents reduced activity. Among the monocyclic analogues, the most potent representatives were five- or six-membered systems with an exocyclic imino group, combined with methyl of ethyl substituents on the endocyclic nitrogen. Introduction of additonal substituents decreased inhibitory potency. 2,3,5,6-Tetrahydro-8H-imidazo[2,1-c][1,4]thiazine and 3-methyl-2-iminothiazolidine have been shown to cause inhibition of lung INMT when administered orally to rabbits.


Synthesis and activities of antitumor agents. N-(2-Chloroethyl)-N-nitrosocarbamoyl derivatives of glycosylamines have been prepared. Six N-(2-chloroethyl)-N-nitrosoureas, including three disaccharide derivatives, were submitted to a determination of antitumor activity. All the compounds tested exhibited strong antitumor activity against leukemia L1210 in mice.


Metabolic oxidation of nicotine to chemically reactive intermediates. Studies on the metabolism of nicotine by rabbit liver microsomal fractions in the presence of 0.01 M sodium cyanide have led to the characterization of two isomeric cyanonicotine compounds. The locations of the cyano groups were established by GC--EIMS analyses of the deuterium-labeled products obtained from the specifically deuterium-labeled substrates (S)-nicotine-5',5'-d2, (R,S)-nicotine-2',5',5'-d3, and (R,S)-nicotine-N-methyl-d3. One cyano adduct was shown to be 5'-cyanonicotine, a product previously isolated from similar microsomal preparations. The second cyano adduct was shown to be N-cyanomethyl)nornicotine; this structure assignment was confirmed by synthesis. Formation of N-cyanomethyl)nornicotine appears to occur, at least in part, without prior nitrogen--carbon bond cleavage, implicating the in situ generation of the N-methyleniminium species during the course of metabolic oxidative N-demethylation of nicotine.


Antiallergy agents. 1. 1,6-Dihydro-6-oxo-2-phenylpyrimidine-5-carboxylic acids and esters. The synthesis of some 1,6-dihydro-6-oxo-2-phenylpyrimidine-5-carboxylic acids and esters with potent oral and intravenous antiallergic activity against passive cutaneous anaphylaxis in the rat is described. Requirements for high activity include a free NH group in the pyrimidinone nucleus and a small to medium size ortho alkoxy or alkenyloxy group on the phenyl ring. It is suggested that in the case of the highly active compounds hydrogen bonding occurs between a nitrogen of the pyrimidine ring and the ethereal oxygen. The nature of this bonding and its possible contribution to an optimum configuration for the molecules is discussed.


New bronchodilators. Synthesis and bronchodilating activity of some 3-(alkoxymethyl)-alpha-(N-substituted aminomethyl)-4-hydroxybenzyl alcohols. A series of 3-(alkoxymethyl)-alpha-(N-substituted aminomethyl)-4-hydroxybenzyl alcohols was synthesized as potential bronchodilators. The ability to prevent effects against histamine-induced bronchoconstriction in guinea pigs was studied to determine their bronchodilating activity. Introduction of a methoxymethyl group in place of the m-hydroxyl group of beta-adrenergic catecholamines afforded compounds especially effective in delaying histamine-induced bronchoconstriction in guinea pigs. Appropriate N-substitution also enhanced the potency of these catecholamine analogues. 4-Hydroxy-3-(methoxymethyl)-alpha-[N-[4-(methoxymethyl)-alpha-methylphenyl]aminoethyl]benzyl alcohol hemifumarate (3r) was the most potent compound in this series.


Studies on antianaphylactic agents. 6. Synthesis of some metabolites of 6-ethyl-3-(1H-tetrazol-5-yl)chromone and their analogues. The metabolites of 6-ethyl-3-(1H-tetrazol-5-yl)chromone (AA-344) (1), an orally effective antiallergic agent, and their analogues were synthesized to confirm the proposed structures and to determine their activity in the rat passive cutaneous anaphylaxis (PCA) test. A glucuronic acid metabolite (6) was assigned the structure 24b, 1-deoxy-1-[5-(6-ethylchromon-3-yl)tetrazol-1-yl]-beta-D-glucopyranuronate, by the comparison of 13C NMR, mass spectra, and TLC of isomeric compounds. In 13C NMR spectra, the shift difference of the tetrazole ring carbons between a pair of isomers was more remarkable than that of the glycosidic carbons. Therefore, the former is a useful criterion for distinguishing between such isomers. Some of the metabolities and analogues were active when administered intravenously, and two metabolites (2 and 3) were also effective upon oral administration.


2-Mercaptoacetamidines as gastric antisecretory agents. A series of N-substituted 2-mercaptoacetamidines was synthesized and evaluated for gastric antisecretory activity in dogs stimulated with gastrin tetrapeptide. The most potent analogues showed 80--95% inhibition of acid secretion after an oral dose of 8 mg/kg. Thus, these compounds represent a new structural type having significant antisecretory activity. Disulfides had essentially the same antisecretory potency as the corresponding mercaptoacetamidines, indicating a metabolic interconversion. Alkylation of the mercapto group decreased potency. Higher carboxamidine homologues such as 2- and 3-mercaptopropionamidines had very low activity. Hydroxyacetamidines and mercaptoacetamides also had low potency. Side effects observed with this series of compounds included emesis, tachycardia, and gastric bleeding.


Preparation and antidiabetic activity of some sulfonylurea derivatives of 3,5-disubstituted pyrazoles. Four series of p-[3,5-dimethyl- (and 5-methyl-3-carboxy-) pyrazole-1]benzenesulfonylureas, thioureas, 2-thiohydantions, and 5,6-dihydro-4(3H)-oxo-2(1H)-pyrimidinethiones were prepared for evaluation as hypoglycemic agents. Biological testing of these compounds showed that some possessed antidiabetic activity.


Differential solubilities in subregions of the membrane: a nonsteric mechanism of drug specificity. We studied the effect of four volatile anesthetics and convulsants on the GABA- and glutamate-induced conductance change in crab muscle. The intensity of drug action correlated with the solubility parameter (delta) values of the four drugs. Thus, the higher that value was for a given drug, the stronger was its effect on the glutamate response but the weaker was it on the response to gamma-aminobutyric acid (GABA). We suggest that different gating molecules are housed in specific subregions of the membrane, each characterized by a particular value of the solubility parameter. The differential distribution of drugs in these subregions may be a nonsteric mechanism for drug specificity.


Conformations, DNA binding parameters, and antileukemic activity of certain cytotoxic protoberberine alkaloids. The tetrahydroprotoberberine alkaloids 5 and 7 possessing a trans-quinolizidine conformation display in vitro KB cytotoxicities in contrast to their corresponding diastereomers 4 and 6 which exist in the cis-quinolizidine conformation and are much less toxic. The DNA-binding parameters of these compounds as well as the protoberberine salts 1, 8, and 9 have been examined by equilibrium dialysis. Only the quaternary salts bind to DNA. The alcohol 5 showed low in vivo activity against leukemia P388 systems, while the quaternary salts 8 and 9 proved to be toxic to the host.


Synthesis and pharmacological properties of [5-isoleucine]-, [8-isoleucine]-, and [5,8-diisoleucine]bradykinin. Three bradykinin analogues have been synthesized in which the phenylalanine residue(s) at positions 5 and/or 8 have been substituted by isoleucine. All these analogues have weak bradykinin-like activity in isolated rat uterine smooth muscle or in rat blood pressure assay. No antagonistic activity was observed with any of these analogues. The importance of phenylalanine at positions 5 and 8 is discussed.


A study of faculty attitudes toward Ohio State's three-year medical program. Three-year curricula, once a proliferating innovation in medical education, are now rapidly waning with the fading of capitation support and the easing of physician manpower shortages. In this article the authors examine faculty opinion of a three-year medical program at the Ohio State University. Using a Likert attitude-scaling technique, the authors developed a questionnaire and administered it to full-time faculty members. Five issues upon which faculty opinion was found to be most diverse include: adequacy of content in a three-year program, time available for faculty to do research, work demands on faculty, basic medical knowledge of three-year students, and quality of post-M.D. positions obtained by three-year graduates. Overall, three general deficiencies in the three-year curriculum, as perceived by faculty, emerged: students have limited instruction in basic medical knowledge and skills; unnecessary emotional, physical, and economic demands are imposed; and neither student learning nor quality of teaching is enhanced.


Arizona's three-year medical curriculum: a postmortem. The College of Medicine at the University of Arizona was among many medical schools instituting a course of study leading to graduation in three years. Students and faculty found the three-year program to be an unsatisfactory educational experience. Among the major problems were excessive intensity of classroom work, insufficient vacation time, and inadequate time to teach important basic science material. Objective measures of student performance demonstrated no differences between graduates of the new program and students graduating from a traditional four-year curriculum. A process of curriculum review involving students, faculty, and administrators ultimately resulted in several curricular improvements and establishment of a new four-year program.


Medical schools and public health departments: a new alliance for progress. For some time there has been concern regarding the future and relevance of both public health departments and departments of community medicine. Problems have developed as to how these agencies can be in the main stream of the health delivery system and maintain quality in service, research, and teaching. In this paper the authors describe an experimental health care delivery and teaching model, the health-department/medical-school affiliation. They analyze the conceptual basis for the affiliation and discuss the critical items in the affiliation agreement in the belief that such an arrangement could have an impact comparable with that which the medical-school/teaching-hospital affiliation had 40 years ago. The options opened by the new affiliation should serve to release the full potential of community medicine and public health in the United States.


A comparison of two approaches to producing competent primary care physicians. The current shortage of primary care physicians demands that medical schools implement procedures to ameliorate this deficiency. Two general approaches to the problem were evaluated in regard to their utility as possible solutions. It was concluded that only an approach that advocates medical curriculum manipulation offers a viable direction for medical educators. Conversely, a technique that focuses primarily on the selection of medical school applicants possessing certain biographical characteristics was found to have a number of inherent shortcomings that negate its effectiveness.


Cardiopulmonary disease in newborns: a study in continuing medical education. A film emphasizing the importance of tachypnea as an early manifestation of congenital heart disease was shown to physicians and nurses at 27 hospitals as part of their regular continuing medical education activities. To evaluate the effects of the program, investigators developed a pretest-posttest design which included a nonequivalent control group. Pretest and posttest data were obtained through chart audit of referrals from subjects in experimental and control groups. Dependent variables used to test the hypothesis included the age at which infants were referred and the age at which tachypnea was noted. Analysis of the data yielded significant gain scores for the experimental group, while changes in the control group were not significant. The findings indicate that a need-oriented educational program can have a measurable impact on improving the quality of patient care.


Application of cluster analysis for characterization of spatial distribution of particles by stereological methods. A method for the detection and characterization of clusters of particles observed in section with the electron microscope is presented. Cluster analysis is performed by the division method described by Berthet et al. (1976). Starting from a single cluster, profiles from each electron micrograph are successively classified in sets containing an increasing number of clusters. The decrease in the mean free distance, lambda, between profiles in the clusters, is used for terminating the subdivision procedure. The function relating the mean free distance with the number of clusters is evaluated in each subdivision set. The actual number of clusters is selected on the basis of the slope of that function, at a point where lambda has a value close to the average profile diameter. The method assumes a convex shape for the clusters; the salient feature is that it provides a physical delineation of clusters in the section. Hence, an evaluation of some characteristics of clusters in the three-dimensional sample may be obtained by using standard stereological procedures. Characterization of the volume to which the individual particles of a population are eventually restricted can as a result be performed. Practical problems in the acquisition of the data needed for cluster analysis are discussed and a system using for that purpose a Quantimet 720 image analyser in a basic configuration, connected on line with a PDP 11/10 minicomputer, is presented. Application of the method is illustrated by the analysis of lysosomes in cultured hepatoma (HTC) cells, at the end of mitosis and during the S phase. Cluster analysis shows that in cells actively synthesizing DNA they are grouped in clusters representing 5.7% of the cellular volume. Moreover, the average number of particles per cluster falls from a minimum of thirteen at mitosis to only six at the S phase.


The construction of a membrane slide support for the observation of living cells. The construction of a slide for examining living cells under the microscope under controlled conditions is described.


A new inexpensive specimen carrier for freeze-fracturing. This paper describes the production and use of a new specimen carrier for the Balzers freeze-fracturing unit. The carriers are inexpensive, easily produced and have excellent mechanical and thermal properties. The rectangular form of the carriers provides easy handling and unequivocal orientation of anisotropic specimens.


Determination of membrane thickness distribution from orthogonal intercepts. This report deals with the reconstruction of the distribution of membrane thickness T from that of orthogonal length Lo, measured in random section planes. In such planes the membrane appears as a band and the linear distance from one of its boundaries perpendicular to the opposite one is the length of the orthogonal intercept. Using a membrane model, an integral equation relating the probability density functions of orthogonal intercept length f(lo) and membrane thickness g(tau) is derived. Relations between moments are derived and the analytic solution to the problem of reconstructing g(tau) from f(lo) is given. The parametric approach by which it assumed that g(tau) has some known analytic form with unknown parameters is considered, and the use of a suggested analytic form for describing the thickness distribution of the human glomerular basement membrane is discussed.


Scanning electron and light microscope correlation of individual human bone marrow cells before and after culture in nutrient agar. This study was undertaken with the aim of identifying the different cell types found in human bone marrow by examining their surface morphology. In an attempt to obtain a homogeneous cell population, cells were both fractionated by discontinuous albumin density gradient centrifugation (DADGC) and selectively grown in nutrient agar. Both cell preparations underwent the critical point drying technique before examination under both the scanning electron microscope (SEM) and subsequently the light microscope (LM). When the SEM image of individual cells was compared with the corresponding LM image, it was not easy to identify the different cell types, because of the shrinkage and distortion that occurred during their preparation. The shrinkage observed under the SEM amounted to a 45% reduction in mean cell diameter. This shrinkage was confirmed by comparing the SEM and LM images of the same cell. Although shrinkage occurred throughout the dehydration sequence, critical point drying was responsible for a 25% reduction in mean cell diameter. Furthermore, direct observation under LM of fixed cells drying in air from ethanol, revealed visible contraction of the cell and distortion of the cell membrane. We assume that a similar morphological change occurred during critical point drying. We conclude that the shrinkage and distortion, caused by the dehydration process involved in SEM preparation, severely limit the value of a study of surface morphology by SEM in the identification of the different cell types found in human bone marrow.


Freeze substitution for preservation of ciliated surfaces for scanning electron microscopy. A technique is described for arresting rapid movement of living cells and preserving their fine surface structures for scanning electron microscopy. Rapid freezing is recommended as the method of immobilization and freeze substitution has been employed to fix and dehydrate the specimens; this technique is more reliable than osmium fixation, both in terms of obtaining a much higher proportion of good results and in the improved preservation of detail. Various techniques of substitution have been investigated for best preservation, and the roles of the constituents of the substitution mixture have been discussed.


Functional significance of the mandibular symphysis. The purpose of this investigation was to relate the morphology of connective tissues in the mandibular symphysis to the behavioral and experimental evidence for mobility and mechanical stress at the symphysis. The anatomy of the symphysis was examined histologically in 6 mammalian orders encompassing 22 species. Behavioral and experimental evidence of stress during the power stroke of the chewing cycle correspond with stresses at the symphysis implied by the location and orientation of symphyseal connective tissues. These stresses are: (1) dorsoventral shear of the symphysis due to the transfer of force from balancing to chewing sides, (2) bending of the symphysis causing tension along the inferior and compression along superior borders due to torsion on the dentaries from the jaw closing muscles, and (3) antero-posterior shear of the symphysis due to an anteriorly directed stress on the chewing side. Interspecific comparisons suggest that leaf eaters can resist greater dorsoventral shear than fruit or insect eaters, but no correlations exist between diet and bending or antero-posterior shear. This suggests that chewing leaves requires larger biting forces.


Morphology and distribution of type II supraependymal cells in the third ventricle of the guinea pig. The distribution and morphology of phagocytic (Type II) supraependymal cells residing within the third ventricle of the guinea pig were investigated by scanning electron microscopy. Type II supraependymal cells were restricted to nonciliated regions of the ventricle. They were most numerous on the choroid plexus, abundant within the infundibular recess and were present on the ventricular floor in the region of the median eminence. Morphologically, they were characterized by a soma from which pseudopodia-like processes extended to the subjacent ependyma. Type II cells varied in configuration according to their location. Those residing on the choroid plexus typically had irregular somas and possessed processes that generally terminated in finger-like extensions. In contrast, cells on the ventricular floor and within the infundibular recess were stellate and possessed processes that terminated in fan-like cytoplasmic expansion. There were no differences noted in the frequency, distribution or morphology of Type II supraependymal cells in male and female animals. Furthermore, cell frequency did not appear to vary in relation to the estrous cycle. The data suggest that the pleomorphism exhibited by Type II supraependymal cells may reflect adaptations to diverse environmental conditions present within different regions of the third ventricle.


The interface between premedicine and medicine: expectations of premedical advisors. Premedical advisors can carry out many varied activities, but all require commitment, resources, and personal interest. Premedical and health career advising appear to be most successful when the task is not assigned to one individual, but is assumed as a university responsibility. Also, premedical advisors seem to be valued more and to have fewer conflicts when they are known by health professional schools and work together with them. Medical schools have a responsibility to assist and support premedical advisors more than is done at present. This need will become increasingly important if the high quality of applicants to medical school is to be maintained in an apparently shrinking applicant pool.(1)


Shigellosis: analysis of 176 isolations. Shigellosis, a worldwide epidemiological problem, is caused usually by six serotypes of Shigella. Man is the principal host, often infected by person-to-person contact. The number of reported cases of shigellosis has progressively increased during the past decade. Because of the observation, the authors reviewed all isolations of Shigella species in patients at Howard University Hospital. There were 176 isolations from 53 patients during the 4½ year period of the study. The study emphasizes the scope and distribution of the disease and the effectiveness of isolation and identification procedures.


Laryngeal tuberculosis: a case report.

Laryngeal tuberculosis: a case report. A case of laryngeal tuberculosis, in a 21-year-old female, is presented. The pathophysiology and natural course of the disease are discussed. The incidence of apical tuberculosis and associated laryngeal spread is noted.


Lung abscess: a review of three-years' experience at the University College Hospital, Ibadan. The experience with 45 patients with lung abscess over a three-year period at the University College Hospital (UCH), Ibadan, is presented. This study confirms the rarity of this disease among Nigerian children and its prevalence in young adults in the third and fourth decades of life. The most common presenting symptoms were purulent cough, chest pain, fever, and life-threatening hemoptysis which was the sole indication for emergency operation in 14 out of 16 patients who were treated surgically. The predominance of these abscesses in the right lung, especially in the superior segment of the lower lobe, supports the fact that aspiration of infected material, following depressed level of consciousness, esophageal obstruction, foreign bodies, and oral sepsis form the major causative factors in patients with lung abscess. The frequent association of sickle cell disease, bronchiectasis, hypertension, and pulmonary aspergilloma contribute significantly to the morbidity and mortality attendant to this disease in our environment. Twenty-nine patients were treated medically with five deaths and 16 patients were treated surgically with six deaths. The high operative mortality (37.5 percent) in this series was due to the extreme emergency conditions under which these patients were operated.


Aseptic necrosis of the hip in sickle cell disease. Aseptic necrosis of the hip occurs in people with sickle cell disease. No one physician has had a great deal of experience treating this lesion. Because sickle cell patients are felt to have decreased longevity, traditional treatment is frequently not offered or considered. The purpose of this paper is to report the results in patients using a more conservative approach.


Spontaneously disappearing gallstones.

Spontaneously disappearing gallstones. A case of spontaneously disappearing gallstones, in a patient who had no evidence of a biliary fistula or gallbladder surgery, is presented.


Renal hamartoma (angiomyolipoma) and the tuberous sclerosis complex. Renal hamartoma is found in 40 to 80 percent of patients with tuberous sclerosis. Microscopic demonstration of fat in the tissues of the mass is felt to be the most reliable diagnostic criterion of hamartoma.Characteristically, the angiographic appearance demonstrates a large, dilated feeding vessel passing through the mass with multiple, multisacculated aneurysmal dilatations appearing like bunches of grapes. There is a delicate neovascularity without A-V shunting and an onion-peel or whorl-like appearance in the venous phase.This case is presented to point out the close association of renal hamartoma and tuberous sclerosis and the need to search for renal hamartoma when the diagnosis of tuberous sclerosis is made.


Topical versus systemic antibiotics in the treatment of acute superficial skin infections. Use of antibiotics in some superficial skin infections is examined. The choice of a route of administration is dependent on a number of factors, including the site and extent of skin lesions, frequency of recurrence, and clinical and immunological state of the host. However, the consensus of various studies on the subject seem to indicate a preference for the systemic route in acute infectious dermatoses.


Non-infantile intussusception.

Non-infantile intussusception. In Europe and North America, most cases of non-infantile intussusception are associated with intestinal neoplasm. At the University College Hospital, Ibadan, Nigeria, the majority of cases of intussusception seen are ileocecocolic and cecocolic types. These are usually not associated with intestinal neoplasm and simple reduction is all that is required.Intussusception occurring in other segments of the intestinal tract is found to be associated with intestinal neoplasm, mesenteric lymphadenopathy, and ascaris worms. The very mobile cecum and ascending colon found in this population may be related to the high incidence of ileocecocolic and cecocolic intussusception.Indications for resection of intussusception include presence of intestinal neoplasm, irreducibility, gangrenous bowel, and chronicity.


Long vs short-course antibiotic prophylaxis in cesarean section: a comparative clinical study. A comparative clinical trial of short-course and long-course ampicillin prophylaxis in cesarean section was completed at Ife University Teaching Hospitals Complex. The results demonstrate a decided advantage in using the short-course regimen in the various parameters measured. The study constitutes a significant advance in efforts to cut medical costs.


Management of obesity in black females in a community model clinic: A preliminary study. During a three-month period, March-May 1978, 23 black females were accepted as patients in the obesity program at the model office of the Department of Community Health and Family Practice, College of Medicine, Howard University.An aggregate of 55 visits made by these patients was recorded for the same period.The mean age of the patients was 38.3 years (SD:13.67), and half of the patients were below 40. Nearly 70 percent had a history of obesity in either one or both parents. More than one half had had high blood pressure and 70 percent of the patients had attempted dieting previously. Common snacks taken by the patients were carbohydrate-rich foods.Based on previous medical and dietary history and behavior patterns related to food intake, the patients were prescribed low-calorie or modified high-protein diets. Each patient at the clinic had a program devised individually for her. A team consisting of a physician, nutritionist, and health educator looked for patterns of behavior causing patients to overeat.The mean initial weight of all patients was 218 lb at registration and the weight after seven weeks of follow-up was 213.8 lb.


Decelerating the aging process.

Decelerating the aging process. Diseases whose incidence and prevalence are increased in the elderly and whose cytopathology, hormones, and immunogenesis differ, generally are included in the field of geriatrics. These conditions may be precipitated or accelerated in quantity or type by a wide variety of genetic and environmental factors. Chronological and progressive deterioration of selected cells, organs, and tissues, and their functions may occur without major specific pathology. These processes are referred to as senescence and its study, is gerontology.Geriatrics includes senility and diseases of the elderly. Terms associated with gerontology include benign agism or senescence, as a normal consequence of the aging processes common to all biological forms of life.The age period, 65 to 85 years, does not necessarily imply senility, but the normal chronological aging of an individual in an industrialized, urbanized society. This paper emphasizes recommendations for deceleration of the normal aging process.


Evaluation of compression spot films in duodenal cap ulcers: study of 63 cases. Eight hundred and twenty patients were examined in an effort to test the abilities of compression studies, erect spot film studies, and air contrast examinations to detect duodenal cap ulcers. The 63 ulcers found are reviewed.


Thyroid gland disorders in African children. Although endocrine disorders are uncommon in black African children, thyroid gland disorders made up 37 percent of all types of 27 endocrine disorders seen at University College Hospital, Ibadan, Nigeria over a five-year period. The age range of the children was four months to 14 years. Diagnoses largely depended on clinical acumen and a few simple laboratory tests. Even the patients with congenital thyroid gland disorders tended to present late with florid classical clinical features, including disturbances of physical and mental development.


Rationale for a program in community ophthalmology. The high incidence of eye abnormalities among blacks, coupled with the relative unavailability of ophthalmic services, has resulted in excessive rates of blindness otherwise preventable or curable. As a consequence, incidence rates for blindness are higher for the black population than the white population.Statistics show that black children requiring eyeglasses are less likely to receive eyeglasses than similar age-matched white children. In virtually every state surveyed, blacks were more frequently and severely afflicted with eye diseases and were less likely to receive treatment than whites. Yet, no programs exist in any state specifically targeted to promote eye care among blacks and reverse the ever-worsening trend. Additional data characterizing the scope of the problem shall be presented.An initial strategy for approaching the problem, namely, community ophthalmology, has been developed. Community ophthalmology represents a new discipline promoting eye health and blindness prevention through programs utilizing methodologies of public health, community medicine, and ophthalmology. Basically, the use of community ophthalmology strategies is critical to positive out-come, when given a population composed of blacks and other minorities who are chronically underserved with regard to all sectors of health.The evolution and rationale for these concepts are discussed from national, as well as international, perspectives.


Particle radiation therapy: requiem or reveille. The 1960s and 1970s witnessed a surge of many institutions devoted to electron therapy. Currently, many facilities are adding or have added particle types of radiation to their armamentarium against cancer. The authors review the concepts, problems, and potentials of this form of therapy.


The child with precocious puberty.

The child with precocious puberty. Precocious puberty can be very confusing and frightening to an affected child and his or her family. The following presentation includes the pertinent physiology, psychology, etiology, diagnosis, and treatment of precocious pubertal development. Radioimmunoassays for gonadotropins and several key hormones are now available which enable the clinician to evaluate and follow the patient's condition with confidence and expertise. Three drugs have been proven effective and the most recent one, cyproterone acetate, has produced exciting clinical remissions with virtually no known side effects.


Herpes simplex in gynecology and obstetrics. The problems of herpes simplex in gynecology and obstetrics are reviewed. There were 40 patients showing the characteristic cellular changes of herpetic infection among the 79,357 Papanicolaou smears taken. Four patients, with cytologic evidence of herpes virus, also had cervical dysplasia. The relationship between herpes virus and dysplasia, carcinoma in situ, and invasive carcinoma is discussed. The use of a properly timed Papanicolaou smear to alert the physician to the danger of an infected birth canal is described. Three cases of neonatal herpes are cited.


Administration of cyclophosphamide during late pregnancy and early lactation: a case report. Burkitt lymphoma was diagnosed in a patient during the third trimester of pregnancy. Treatment with a low-dose, seven-day course of intravenous cyclophosphamide gave a good response which permitted the pregnancy to be carried to term, with delivery of a normal infant. When the mother received cyclophosphamide during lactation while the baby was breast-fed, the infant's leukocyte and platelet counts were rapidly depressed. Results in this patient and a survey of reported cases in which cyclophosphamide was administered during pregnancy and lactation lead to the conclusions that (1) low-dose intravenous cyclophosphamide therapy is not hazardous to the fetus during late pregnancy; (2) mammary concentration of the drug is too toxic to the infant's bone marrow; and (3) breast-feeding the baby should be suspended during the period the mother is receiving cyclophosphamide treatment.


Hand-foot roentgen findings in sickle cell anemia. X-ray findings in the hands and feet of 420 sickle cell anemia patients were studied during the six-year period, 1972-1978. A correlation between roentgen findings and clinical presentations is emphasized.


Evaluation of an academic reinforcement program for health science students. An Academic Reinforcement Program has been used for three years at the University of Texas Medical Branch to assist health science students with test taking, note taking, memorization, labeling, charting, and time-scheduling techniques. An evaluation indicated that the majority of students who have learned the techniques improved their academic grades with less expenditure of study time, learned to deal with some problems which affected their studies, and gained self-confidence through the encouragement and support of the staff who taught the techniques.


Dental health problems of the geriatric population. A topic seldom considered and even more rarely discussed among the dental components of the health professions is presented. The dentist's preoccupation with hosts of dental treatment problems has left little time for considering the perplexities of aging, critical illness, or dying or dead patient's relatives. Community dentistry has now forced upon our consciousness the fact that perhaps the subject is one which must not be avoided, if we are to fulfill completely our professional obligations to the public.


Black contributions to the early history of Western medicine: lack of recognition as a cause of black under-representation in US medical schools. During several millenia, blacks in ancient Egypt made numerous contributions to medicine and were acknowledged as the inventors of the art of medicine. They produced the earliest physicians, medical knowledge, and medical literature. They contributed to the development of medicine in ancient Greece. Ancient writers, including Herodotus, Isocrates, and Diodorus, affirm this. Modern presentations of ancient medicine, however, deprive blacks of the knowledge of their early contributions to medicine by ignoring or subtly misrepresenting the black identity of the ancient Egyptians. Blacks are currently under-represented in US medical schools. It is proposed that the recognition of the contributions of blacks to the early history of Western medicine would inspire black students to study medicine.


NCTR/NCI symposium on the use of inbred and outbred animals in toxicological testing: objectives of the conference. The purpose of this conference of toxicologists, geneticists, and statisticians is to increase understanding of the role of animal genetics in toxicology by discussion of one aspect of this relationship, namely the genetic structures of the laboratory animal populations used in toxicological assays. It is hoped that discussion and comparison of various attributes of inbred, F1 hybrid, and outbred laboratory animal populations will result in recommendations concerning the most appropriate genetic population structures to be used to supply test animals for toxicological studies having different objectives and end points.


Influence of genetic population structure on the results of chronic toxicity studies. The importance of the genetic makeup of animals used for toxicological testing has been recognized, although there are few data regarding this. In the study discussed here offspring from a BALB/cStCrlfC3Hf/Nctr male and C57BL/6jfC3Hf/Nctr female cross were used to produce a homogeneous (F1) strain, and brother-sister mating of the F1 population was used to produce a heterogeneous (F2) strain of mice, both having essentially the same gene pool but with different distributions. The end points of mortality and body weights were used to demonstrate relative variability about a mean resulting from genetic constitution when the mice were exposed to benzidine dihydrochloride. This life-span study demonstrated that genetic constitution in research animals should be an important consideration in toxicological research. There were significant differences in body weight and mortality between the two populations, with the F2 mice exhibiting wider body weight ranges and greater susceptibility to the test chemical.


Influence of genetic variables on means, variances, and covariances in behavioral responses to toxicological and pharmacological substances. Inbred strains and F1 generations provide strong experimental control in toxicological studies by virtue of the relative stability of their means. Their variances do not always conform to simple expectations, with respect either to each other or to segregating populations. For the evaluation of covariances or correlations, populations of choice are genetically segregating ones. A genetic approach of demonstrated power but as yet limited application to toxicological problems is that of selective breeding, a procedure by which lines of animals can be developed to precise specifications.


Genetic and phenotypic consequences of inbreeding. The effects of inbreeding on heterozygosity, mean performance, variation within and between lines, and fixation or loss of genes are discussed. Theoretical predictions for degree of pedigree inbreeding and consequences for genes neutral with respect to fitness are outlined first, and then the likely modifications necessary to account for natural selection during inbred line development are considered. For illustration, some results from experiments with laboratory animals are reviewed. Inbred lines or F1 crosses between them are special genotypes, not representative of the population from which they were derived. Similarly, outbred stocks of laboratory animals that have a long history in the laboratory, or narrow base, or are maintained in small numbers, will lack variation and become like inbred lines. The efficacy of using either inbreds or such outbred stocks in toxicological screening is questioned on the ground that the range of genotypes is inadequate.


Genetic constitution and response to toxic chemicals--an overview. There is no scientific justification for the use of random-bred or outbred rodents in toxicological and carcinogenesis testing. The frequent desire for heterozygosity in a test population can best be met by using F1 hybrids of inbred progenitor strains. Extrapolation of animal data to humans will ultimately come through an understanding of cellular responses to carcinogenic and toxicological agents. Isogenic animal populations such as inbred strains, F1 hybrids, and the recently developed recombinant inbred lines are currently our best approach to resolving these mechanisms.


Properties of inbred strains and outbred stocks, with special reference to toxicity testing. Genetically uniform or isogenic strains (inbred strains and F1 hybrids) have a number of properties that make them valuable in toxicity testing, provided the experimental design can be modified to include more than one such strain. Isogenicity and homozygosity lead to great phenotypic uniformity, high long-term genetic stability, high identifiability, large differences between strains (individuality), and the possibility of setting up daughter colonies genetically identical to the parents. This in turn means that many isogenic strains are internationally distributed, and that considerable background data exist on the characteristics of the common strains. Toxicity testing usually involves the calculation of dose-response curves. Use of phenotypically variable nonisogenic stocks reduces the precision with which such curves can be estimated, without many other benefits. A single isogenic strain may not be satisfactory as it may be resistant to the drug being tested. However, the use of several isogenic strains with a factorial experimental design would overcome this problem, would give high statistical precision, and would provide a better basis for extrapolation to humans than the use of a single stock. Such a design would make it possible to choose a range of strains on the basis of known drug sensitivities, it would be highly repeatable, it would allow comparison of different drugs, and it would show whether the response was genetically determined. The benefits of such an experimental design far outweigh the disadvantages, which are mostly of a practical nature.


Genetic aspects of metabolic processing of chemical substances. The Ah locus in the mouse controls the induction of cytochrome P1-450 and at least 10 associated monooxygenase activities. These enzyme systems metabolically potentiate and detoxify chemical carcinogens, environmental pollutants, drugs, and other chemicals, as well as numerous endogenous substrates. With certain substrates cytochrome P1-450 is known to produce a predominance of reactive intermediates and products that differ from those formed by other cytochrome P-450 species. Numerous conditions in the mouse, including cancer, drug toxicity, and birth defects, are directly associated with the Ah locus, which is considered to be a single gene or a small number of genes.


Statistical design of toxicity assays: role of genetic structure of test animal population. This paper concerns certain statistical aspects of the problem of among-strain differences in cancer susceptibility and how these differences may affect the design of toxicity assays. First, the data of Innes et al. (1969) were examined to investigate the magnitude of within-study, between-strain differences in tumor induction. Although there was a very high overall association between mouse strains with respect to the induction of hepatomas, evidence of strain-to-strain variability was found for several compounds. Next, a number of long-term carcinogenicity studies with DDT were considered, and among-strain differences in cancer susceptibility for this compound were noted. Finally, it was shown that if susceptible subgroups do exist, and certain simplifying assumptions are made, then in many cases tumor increases can be detected more readily by studying several inbred mouse strains rather than a single outbred stock.


Current problems in the choice of animals for toxicity testing. Animal models for the study of toxicity are chosen more for pragmatic reasons such as life-span, ease of handling, and economics than because of a comprehensive process of validation. There is also a tendency to seek the answers to nonspecific questions and then to use those answers in situations where they cannot lead to proper judgments. In some cases, such as in carcinogenicity testing, an uneasy compromise exists between the sensitivity of the model and the background noise. There is scope, by increased attention to both the control of environmental factors and the genetics of the animal models, to increase the sensitivity and validity of the systems that are used in toxicology.


Coding of increments and decrements in stimulus intensity in single units in the cochlear nucleus of the rat. The response of single units in the cochlear nucleus when confronted with step increments and step decrements in stimulus intensity was studied in the rat using tones that were amplitude modulated with square waves or with pseudorandom noise. Cycle histograms of the responses to tones modulated with square waves revealed that the probability of firing increased as a result of step increments in stimulus intensity for tones at characteristic frequency (CF) and that the probability of firing decreased as a result of step decrements. When two tones were presented simultaneously, one at CF and one at the unit's best inhibitory frequency (BIF), and one or the other of the tones was modulated, the modulation of the cycle histogram of the responses was greater than when only one tone was presented. Modulation of the inhibitory tone gave rise to histograms that were mirror images of the histograms of the responses to modulation of the excitatory tone. An increase in probability of firing always occurred at a faster rate than a decrease, independent of whether the increase was brought about by increase in the intensity of the excitatory tone or by a decrease in the intensity of the inhibitory tone. The cycle histograms of the responses to square wave-modulated tones and the step response estimated from the responses to tones amplitude-modulated with pseudorandom noise showed a greater similarity when the cycle histograms depicted the case of an increase in the probability of firing than when they concerned a decrease in the probability of firing.


Molecular forms of cholinesterases in cerebrospinal fluid, blood plasma, and brain tissue of the beagle dog. In the present study we have investigated the activity of AChE and BuChE in the cisternal CSF of the Beagle dog and have outlined the characteristics of the molecular forms of both enzymes. The same enzymes were also investigated in samples from blood plasma and brain tissue. It is concluded that AChE in CSF originates from the spinal cord and brain tissues as a result of some secretory process. A combined origin from both blood plasma and brain tissue appears to be probable for the BuChE of the CSF.