An examination of John Fewster's role in the discovery of smallpox vaccination.

Edward Jenner is recognised today as the father of vaccination but, as this paper explores, he was not the only Gloucestershire doctor to be linked to this discovery. John Fewster, a local surgeon and apothecary, is also said to have experimented with vaccination, many years before Jenner. This claim is made in a letter addressed to John Coakley Lettsom, written by John Player, a Quaker farmer. Player describes in detail Fewster's realisation that cowpox could be used to protect against smallpox. This letter is frequently cited but has not previously been subjected to critical analysis. We have identified several inconsistencies, including conflicting dates and a possible ulterior motive in that Player's son was to marry Fewster's daughter. We think it unlikely that Player, a devout Quaker, would have consciously fabricated evidence, but argue that the discrepancies in his account undermine the assumption that Fewster carried out vaccination experiments prior to Jenner. We also explore the assertion that Fewster presented a paper in 1765 on the subject of cowpox and its protective effect over smallpox. We conclude that, although there is no doubt that Fewster did pre-empt Jenner's discovery of vaccination, he did not realise the significance or importance of this momentous medical advance.

Influence of breed and genotype on the onset and distribution of infectivity and disease-associated prion protein in sheep following oral infection with the bovine spongiform encephalopathy agent.

The onset and distribution of infectivity and disease-specific prion protein (PrP(d)) accumulation was studied in Romney and Suffolk sheep of the ARQ/ARQ, ARQ/ARR and ARR/ARR prion protein gene (Prnp) genotypes (where A stands for alanine, R for arginine and Q for glutamine at codons 136, 154 and 171 of PrP), following experimental oral infection with cattle-derived bovine spongiform encephalopathy (BSE) agent. Groups of sheep were killed at regular intervals and a wide range of tissues taken for mouse bioassay or immunohistochemistry (IHC), or both. Bioassay results for infectivity were mostly coincident with those of PrP(d) detection by IHC both in terms of tissues and time post infection. Neither PrP(d) nor infectivity was detected in any tissues of BSE-dosed ARQ/ARR or ARR/ARR sheep or of undosed controls. Moreover, four ARQ/ARQ Suffolk sheep, which were methionine (M)/threonine heterozygous at codon 112 of the Prnp gene, did not show any biological or immunohistochemical evidence of infection, while those homozygous for methionine (MARQ/MARQ) did. In MARQ/MARQ sheep of both breeds, initial PrP(d) accumulation was identified in lymphoreticular system (LRS) tissues followed by the central nervous system (CNS) and enteric nervous system (ENS) and finally by the autonomic nervous system and peripheral nervous system and other organs. Detection of infectivity closely mimicked this sequence. No PrP(d) was observed in the ENS prior to its accumulation in the CNS, suggesting that ENS involvement occurred simultaneously to that of, or followed centrifugal spread from, the CNS. The distribution of PrP(d) within the ENS further suggested a progressive spread from the ileal plexus to other ENS segments via neuronal connections of the gut wall. Differences between the two breeds were noted in terms of involvement of LRS and ENS tissues, with Romney sheep showing a more delayed and less consistent PrP(d) accumulation than Suffolk sheep in such tissues. Whether this accounted for the slight delay (∼5 months) in the appearance of clinical signs in Romney sheep is debatable since by the last scheduled kill before animals reached clinical end point, both breeds showed widespread accumulation and similar magnitudes of PrP(d) accumulation in the brain.

Identical pathogenesis and neuropathological phenotype of scrapie in valine, arginine, glutamine/valine, arginine, glutamine sheep infected experimentally by the oral and conjunctival routes.

The pathogenesis of scrapie in sheep after natural or oral exposure to the infectious agent generally involves the early accumulation of disease-associated prion protein (PrP(d)) in the lymphoreticular system (LRS). This phase is followed by neuroinvasion, for which two routes, ascending neural and haematogenous, have been postulated. The present study reports the use of immunohistochemistry to track the tissue progression of PrP(d) deposition in sheep of a single, highly scrapie-susceptible PrP genotype administered by the oral or conjunctival routes. Regardless of the route of infection, the earliest detection of PrP(d) was in gut- and pharynx-associated LRS tissues. Subsequently, the brain became PrP(d) positive simultaneously with other LRS tissues, but before the spinal cord and peripheral nervous tissues of the enteric, parasympathetic and sympathetic systems. The sites of initial PrP(d) accumulation in the brain were the dorsal motor nucleus of the vagus and the hypothalamus and their related circumventricular organs (the area postrema and the median eminence, respectively). These were the same for both routes of infection. Rapid progression to clinical disease was observed in sheep infected orally or conjunctivally, with definite signs of scrapie recorded at around 6 and 8 months after infection, respectively. Longer incubation periods in sheep infected by the conjunctival route were probably due to them receiving a lower dose than those infected orally. Irrespective of the route of infection, clinically affected sheep showed the same pathological phenotype (PrP(d) profile) and PrP(d) distribution throughout the brain. The identical peripheral and central pathogenesis observed in sheep of both groups suggests early dissemination of the infectious agent in the bloodstream and a common neuroinvasion pathway. The late involvement of the enteric and autonomic nervous system supports a haematogenous route of infection to the brain.

Influence of polymorphisms in the prion protein gene on the pathogenesis and neuropathological phenotype of sheep scrapie after oral infection.

The prion protein gene (Prnp) plays a crucial role in the susceptibility of sheep to scrapie in terms of attack rate and/or incubation period. However, the influence of Prnp on the pathogenesis of the disease, specifically the involvement of tissues of the lymphoreticular system (LRS), pathways of neuroinvasion and neuropathological phenotypes, remains controversial. This study reports the onset and progression of disease-associated prion protein (PrP(d)) accumulation in the LRS and nervous tissues of sheep of six different Prnp genotypes infected by oral administration of the same mixed scrapie brain homogenate. Sheep homozygous for glutamine (Q) at codon 171 of PrP, with either valine (V) or alanine (A) at codon 136 (i.e. VRQ/VRQ, VRQ/ARQ and ARQ/ARQ), showed early and consistent PrP(d) accumulation in LRS tissues of the pharynx and gut. In contrast, LRS involvement was minimal, inconsistent and occurred late in the incubation period in sheep heterozygous for arginine (R) at codon 171 (i.e. VRQ/ARR and ARQ/ARR). Despite this difference, all five groups were susceptible to infection and developed clinical disease, albeit with significantly different incubation periods (shortest in VRQ/VRQ and longest in ARQ/ARR sheep). The remaining group of ARR/ARR homozygous sheep did not show evidence of infection at the end of the experiment or at previous predetermined time points. As for LRS tissues, the sites of initial PrP(d) accumulation in the brain were determined immunohistochemically. These were the same in all susceptible sheep (except for ARR/ARR sheep), regardless of their Prnp genotype which, together with an early and consistent accumulation of PrP(d) in circumventricular organs and a late or inconsistent involvement of the enteric and autonomic nervous system and of the spinal cord, suggests neuroinvasion occurring via the blood. The neuropathological phenotype (PrP(d) profile in the central nervous system) of clinically affected sheep was similar in the three V136 carrier groups, but showed some differences in the two A136 homozygous groups, suggesting a codon 136-driven selection of different strains from the mixture contained in the inoculum. ARQ/ARR sheep showed an irregular distribution of brain PrP(d), contrasting with the more widespread distribution of the other four groups. The results indicate that (1) ARQ/ARR sheep are more susceptible to oral scrapie infection than would be predicted from incidence figures in natural disease, (2) amplification and accumulation of PrP(d) in LRS tissues is host genotype dependent, but does not necessarily have a marked effect on the outcome of the infection and (3) the neuropathological phenotype of scrapie is related to the host genotype, but possibly in combination with the infecting source.

Minimal involvement of the circumventricular organs in the pathogenesis of spontaneously arising and experimentally induced classical bovine spongiform encephalopathy.

In sheep infected experimentally with the bovine spongiform encephalopathy (BSE) agent, amplification of infectivity in peripheral organs during early preclinical stages is thought to contribute to high titres of the agent being detected in blood, with subsequent haematogenous neuroinvasion through the circumventricular organs (CVOs). In contrast, little disease-associated prion protein (PrP(d)) or infectivity is detected in the peripheral tissues of cattle during the preclinical and clinical stages of BSE. The aim of this study was to investigate immunohistochemically the role of haematogenous neuroinvasion in cattle with spontaneously arising and experimentally induced BSE. There was almost complete absence of PrP(d) in the peripheral organs of BSE infected cattle. Additionally, there was minimal involvement of the CVOs during preclinical disease and there was progressive caudorostral accumulation of PrP(d) in the brain. These findings do not support haematogenous neuroinvasion in the bovine disease.

Experimental bovine spongiform encephalopathy: detection of PrP(Sc) in the small intestine relative to exposure dose and age.

European regulations for the control of bovine spongiform encephalopathy (BSE) decree destruction of the intestines from slaughtered cattle, therefore producers have been obliged to import beef casings from countries with a negligible BSE risk. This study applies immunohistochemical and biochemical approaches to investigate the occurrence and distribution of disease-associated prion protein (PrP(Sc)) in the duodenum, jejunum and ileum of cattle orally exposed to a 1 g or 100 g dose of a titrated BSE brainstem homogenate. Samples were derived from animals at various times post exposure. Lymphoid follicles were counted and the frequency of affected follicles recorded. No PrP(Sc) was detected in the duodenum or jejunum of animals exposed to a 1 g dose or in the duodenum of animals receiving a 100 g dose. PrP(Sc) was detected in the lymphoid tissue of the ileum of 1/98 (1.0%) animals receiving the 1 g dose and in the jejunum and ileum of 8/58 (13.8%) and 45/99 (45.5%), respectively, of animals receiving the 100 g dose. The frequency of PrP(Sc)- positive follicles was less than 1.5% per case and biochemical tests appeared less sensitive than immunohistochemistry. The probability of detecting lymphoid follicles in the ileum declined with age and for the 100 g exposure the proportion of positive follicles increased, while the proportion of positive animals decreased with age. Detection of PrP(Sc) in intestinal neural tissue was rare. The results suggest that the jejunum and duodenum of BSE-infected cattle contain considerably less BSE infectivity than the ileum, irrespective of exposure dose. In animals receiving the low exposure dose, as in most natural cases of BSE, the rarity of PrP(Sc) detection compared with high-dose exposure, suggests a very low BSE risk from food products containing the jejunum and duodenum of cattle slaughtered for human consumption.

Bovine spongiform encephalopathy: investigation of phenotypic variation among passive surveillance cases.

Bovine spongiform encephalopathy (BSE) is a prion disease of domesticated cattle, first identified in Great Britain (GB) in 1986. The disease has been characterized by histopathological, immunohistochemical, biochemical and biological properties, which have shown a consistent disease phenotype among cases obtained by passive surveillance. With the advent of active surveillance in 2001, immunological tests for detection of the prion protein revealed some cases with different biochemical characteristics and, in certain instances, differences in pathology that have indicated variant phenotypes and the possibility of agent strain variation. This study examines a case set of 523 bovine brains derived from archived material identified through passive surveillance in GB. All cases conformed to the phenotype of classical BSE (BSE-C) by histopathological, immunohistochemical and biochemical approaches. The analyses consolidated an understanding of BSE-C and, by western blotting, confirmed differentiation from the known atypical BSE cases which exhibit higher or lower molecular masses than BSE-C (BSE-H and BSE-L respectively).

11beta-Hydroxysteroid dehydrogenase expression and activities in bovine granulosa cells and corpora lutea implicate corticosteroids in bovine ovarian physiology.

Cortisol-cortisone metabolism is catalysed by the bi-directional NADP(H)-dependent type 1 11beta-hydroxysteroid dehydrogenase (11betaHSD1) enzyme and the oxidative NAD(+)-dependent type 2 11betaHSD (11betaHSD2). This study related the expression of 11betaHSD1 and 11betaHSD2 enzymes (mRNA and protein) to net 11-ketosteroid reductase and 11beta-dehydrogenase (11beta-DH) activities in bovine follicular granulosa and luteal cells. Granulosa cells were isolated from follicles of < 4, 4-8, > 8 and > 12 mm in diameter in either the follicular or luteal phase of the ovarian cycle. Luteal cells were obtained from corpora lutea (CL) in the early non-pregnant luteal phase. Enzyme expression was assessed by reverse transcription-PCR and western blotting, while enzyme activities were measured over 1 h in cell homogenates using radiometric conversion assays with 100 nM [(3)H]cortisone or [(3)H]cortisol and pyridine dinucleotide cofactors. Irrespective of follicle diameter, the expression of 11betaHSD2 and NAD(+)-dependent oxidation of cortisol predominated in granulosa cells harvested in the follicular phase. In contrast, in granulosa cells obtained from luteal phase follicles and in bovine luteal cells, expression of 11betaHSD1 exceeded that of 11betaHSD2 and the major enzyme activity was NADP(+)-dependent cortisol oxidation. Increasing follicular diameter was associated with progressive increases in expression and activities of 11betaHSD2 and 11betaHSD1 in follicular and luteal phase granulosa cells respectively. In follicular phase granulosa cells from antral follicles < 12 mm, 11betaHSD1 migrated with a molecular mass of 34 kDa, whereas in the dominant follicle, CL and all luteal phase granulosa cells, a second protein band of 68 kDa was consistently detected. In all samples, 11betaHSD2 had a molecular mass of 48 kDa, but in large antral follicles (> 8 mm), there was an additional immunoreactive band at 50 kDa. We conclude that 11betaHSD2 is the predominant functional 11betaHSD enzyme expressed in follicular phase granulosa cells from growing bovine antral follicles. In contrast, in bovine granulosa cells from dominant or luteal phase follicles, and in bovine luteal cells from early non-pregnant CL, 11betaHSD1 is the major glucocorticoid-metabolising enzyme. The increasing levels of cortisol inactivation by the combined NADP(+)- and NAD(+)-dependent 11beta-DH activities suggest a need to restrict cortisol access to corticosteroid receptors in the final stages of follicle development.

Alterations in placental 11 beta-hydroxysteroid dehydrogenase (11 betaHSD) activities and fetal cortisol:cortisone ratios induced by nutritional restriction prior to conception and at defined stages of gestation in ewes.

In the placenta, cortisol is inactivated by NADP(+)- and NAD(+)-dependent isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD). Decreased placental 11betaHSD activities have been implicated in intrauterine growth restriction (IUGR) and fetal programming of adult diseases. The objective of this study was to investigate whether placental 11betaHSD activities and fetal plasma cortisol:cortisone ratios could be affected by nutritional restriction of ewes (70% maintenance diet) throughout gestation, for specific stages of gestation, or prior to mating. Chronic nutritional restriction from day 26 of gestation onwards decreased NAD(+)-dependent 11betaHSD activities by 52 +/- 4% and 45 +/- 6% on days 90 and 135 of gestation respectively. Although the decreases in enzyme activities were associated with fetal IUGR, the cortisol:cortisone ratio in fetal plasma was unaffected by chronic nutritional restriction throughout pregnancy. Nutritional restriction confined to early (days 26-45), mid- (days 46-90) and late gestation (days 91-135), or the 30 days prior to mating, had no significant effect on NAD(+)-dependent, placental 11betaHSD activities, nor was there evidence of IUGR. However, nutritional restriction at each stage of pregnancy and prior to mating was associated with significant decreases in the fetal plasma cortisol:cortisone ratio (3.2 +/- 0.7 in control fetuses; 1.0 to 1.6 in fetuses carried by nutritionally restricted ewes). We conclude that nutritional restriction of pregnant ewes for more than 45 consecutive days can significantly decrease NAD(+)-dependent placental 11betaHSD activities in association with IUGR. While the cortisol:cortisone ratio in fetal plasma is sensitive to relatively acute restriction of nutrient intake, even prior to mating, this ratio does not reflect direct ex vivo measurements of placental 11betaHSD activities.

Expression of 11beta-hydroxysteroid dehydrogenase (11betaHSD) proteins in luteinizing human granulosa-lutein cells.

In a range of tIssues, cortisol is inter-converted with cortisone by 11beta-hydroxysteroid dehydrogenase (11betaHSD). To date, two isoforms of 11betaHSD have been cloned. Previous studies have shown that human granulosa cells express type 2 11betaHSD mRNA during the follicular phase of the ovarian cycle, switching to type 1 11betaHSD mRNA expression as luteinization occurs. However, it is not known whether protein expression, and 11betaHSD enzyme activities reflect this reported pattern of mRNA expression. Hence, the aims of the current study were to investigate the expression and activities of 11betaHSD proteins in luteinizing human granulosa-lutein (hGL) cells. Luteinizing hGL cells were cultured for up to 3 days with enzyme activities (11beta-dehydrogenase (11betaDH) and 11-ketosteroid reductase (11 KSR)) and protein expression (type 1 and type 2 11betaHSD) assessed on each day of culture. In Western blots, an immunopurified type 1 11betaHSD antibody recognized a band of 38 kDa in hGL cells and in human embryonic kidney (HEK) cells stably transfected with human type 1 11betaHSD. The type 2 11betaHSD antibody recognized a band of 48 kDa in HEK cells transfected with human type 2 11betaHSD cDNA but the type 2 protein was not expressed in hGL cells throughout the 3 days of culture. While the expression of type 1 11betaHSD protein increased progressively by 2.7-fold over 3 days as hGL cells luteinized, both 11betaDH and reductase activities declined (by 52.9% and 34.2%; P<0.05) over this same period. Changes in enzyme expression and activity were unaffected by the suppression of ovarian steroid synthesis.

Ovarian modulators of type 1 11beta-hydroxysteroid dehydrogenase (11betaHSD) activity and intra-follicular cortisol:cortisone ratios correlate with the clinical outcome of IVF.

BACKGROUND: Follicular fluid (FF) contains compounds that can modulate NADP(+)-dependent oxidation of cortisol by type 1 11beta-hydroxysteroid dehydrogenase (11betaHSD). The objective of this study was to investigate the relationships between levels of the ovarian modulators of type 1 11betaHSD, intra-follicular cortisol:cortisone ratios and the clinical outcome of IVF cycles. METHODS: A single random sample of FF was aspirated from each of 132 patients undergoing gonadotrophin-stimulated IVF. Components of FF, resolved using C18 column chromatography, were evaluated for effects on NADP(+)-dependent cortisol oxidation in rat kidney homogenates. Intra- follicular steroid concentrations were measured by radioimmunoassays. Clinical pregnancies were confirmed by ultrasonography at 6 weeks post-embryo transfer. RESULTS: Levels of the hydrophilic ovarian 11betaHSD stimuli were significantly lower (P<0.0001) and levels of the hydrophobic ovarian 11betaHSD inhibitors were significantly higher (P<0.002) in conception versus non-conception cycles. Intra-follicular cortisol:cortisone ratios increased with the degree of inhibition of 11betaHSD by the hydrophobic FF fractions. FF obtained from conception cycles had significantly higher cortisol:cortisone ratios than samples from non-conception cycles (12.9+/-0.3 versus 8.5+/-0.2, respectively; P<0.0001). CONCLUSIONS: Conception by IVF is associated with elevated intra-follicular cortisol:cortisone ratios, which reflect low levels of ovarian stimuli and/or high levels of ovarian inhibitors of type 1 11betaHSD.

Ovarian modulators of 11beta-hydroxysteroid dehydrogenase (11betaHSD) activity in follicular fluid from gonadotrophin-stimulated assisted conception cycles.

In the ovary, cortisol-cortisone interconversion is catalysed by isoforms of 11beta-hydroxysteroid dehydrogenase (11betaHSD). The objective of this study was to establish whether human follicular fluid (hFF), obtained after controlled ovarian hyperstimulation, contains paracrine modulators of 11betaHSD activity. Of 274 hFF samples tested for effects in rat kidney homogenates, 206 hFF samples significantly inhibited NADP(+)-dependent oxidation of cortisol within 1 h (by 11-67% of control 11betaHSD activity), whereas 42 hFF samples significantly stimulated 11betaHSD activity (16-210% increase relative to control). Although charcoal-stripping of hFF prevented the inhibition and potentiated the stimulation of NADP(+)-dependent cortisol oxidation in a renal homogenate, effects of individual hFF samples on NADP(+)-dependent cortisol oxidation were independent of intrafollicular progesterone concentrations. Hydrophilic fractions of hFF samples, isolated by C18 column chromatography, stimulated both the NADP(+)-dependent oxidation of cortisol (by 55+/-5%, n=98) and the NADPH-dependent reduction of cortisone (by 86+/-22%, n= 5). In contrast, the hydrophobic fractions of hFF (eluted at 65-85% methanol) inhibited both NADP(+)-dependent 11beta-dehydrogenase and NADPH-dependent 11-ketosteroid reductase activities (by 63+/-2% and 74+/-4%, respectively). None of the C18 column fractions of 50 hFF samples had any significant effect on NAD(+)-dependent 11beta-dehydrogenase activities. The hydrophobic inhibitors of NADP(H)-dependent cortisol-cortisone metabolism did not co-elute with several candidate compounds (prostaglandins E(2) and F(2alpha), cortisol, cortisone, oestradiol, testosterone, progesterone, pregnenolone or cholesterol). Hence, hFF aspirated from women undergoing controlled ovarian hyperstimulation for assisted conception contains both hydrophilic stimuli and hydrophobic inhibitors of glucocorticoid metabolism which appear to be selective for the NADP(H)-dependent, type 1 isoform of 11betaHSD.

Morphologically distinct sperm subpopulations defined by Fourier shape descriptors in fresh ejaculates correlate with variation in boar semen quality following cryopreservation.

This study investigated two hypotheses: 1) that consistent between-boar variation in frozen semen quality exists and is genetically determined, and 2) that morphologically distinct subpopulations of spermatozoa exist within fresh boar ejaculates and that the incidence of these subpopulations is correlated with semen quality following cryopreservation. Five ejaculates were collected from each of 15 boars (5 boars from each of 3 breeds). An objective sperm morphology analyzer used Fourier shape descriptors to describe variation in the morphology of 300 spermatozoa per ejaculate before freezing. Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% glycerol) and 5 straws (0.5 mL) per ejaculate were cryopreserved (to -5 degrees C at 6 degrees C/min, then -5 degrees C to -80 degrees C at 40 degrees C/min). Semen was assessed for percentage of motile cells and motility characteristics (with computer-aided sperm analysis), plasma membrane integrity (SYBR-14 positive), and acrosome integrity (fluorescein-labeled peanut agglutinin positive). Consistent between-boar variability was detected for post-thaw sperm motility (P < .01), membrane integrity (P < .01), acrosome integrity (P < .01), curvilinear velocity (P < .01), straight-line velocity (P < .05), beat cross-frequency (P < .05), and amplitude of lateral head displacement (P < .01). Three morphologically distinct subpopulations of spermatozoa, defined by Fourier descriptors, were detected. The proportion of these subpopulations within the fresh ejaculate correlated with semen quality assessments made following cryopreservation. These findings support the hypothesis that consistent interindividual variation in sperm freezability is genetically determined and may relate to processes that occur during spermatogenesis. Subsequent characterization of these genetic differences between "good" and "poor" freezers may ultimately identify biophysical components of the spermatozoa that are essential for successful cryopreservation.

Novel mutations in the gene encoding ATP-binding cassette 1 in four tangier disease kindreds.

Tangier disease (TD) is an autosomal co-dominant disorder in which homozygotes have a marked deficiency of high density lipoprotein (HDL) cholesterol and, in some cases, peripheral neuropathy and premature coronary heart disease (CHD). Homozygotes are further characterized by cholesteryl ester deposition in various tissues throughout the body, most notably in those of the reticuloendothelial system. Several studies have demonstrated that the excess lipid deposition in TD is due to defective apolipoprotein-mediated efflux of cellular cholesterol and phospholipids. Although much progress has been made in our understanding of the metabolic basis of TD, the precise molecular defect had remained elusive until very recently. By positional cloning methods, we: 1) confirm the assignment of TD to chromosome 9q31, 2) provide evidence that human ATP-binding cassette-1 (hABC-1) maps to a 250 kb region on 9q31, and 3) describe novel deletion, insertion, and missense mutations in the gene encoding hABC-1 in four unrelated TD kindreds. These results establish a causal role for mutations in hABC-1 in TD and indicate that this transporter has a critical function in the regulation of intracellular lipid trafficking that dramatically affects plasma HDL cholesterol levels.

Sources of variation in the morphological characteristics of sperm subpopulations assessed objectively by a novel automated sperm morphology analysis system.

There is evidence that the mammalian ejaculate contains distinct subpopulations of spermatozoa and that the variability among these subpopulations may have adaptive and functional significance. This study investigated the precision, reproducibility and operating characteristics of a novel automated sperm morphology analysis system, the Hobson Morphology package, establishing protocols to investigate boar sperm characteristics. Five ejaculates were collected from each of three boars from different genetic lines: Landrace-Meishan introgression, Sireline Large White and Damline Large White. Five semen smears per ejaculate were stained with haematoxylin and eosin. Two hundred spermatozoa per slide were analysed. No significant differences among slides within an ejaculate were detected for sperm tail length (P = 0.770), head width (P = 0.736) and head length (P = 0.615), indicating that both staining and morphology analysis were precise and reproducible. Among the boars, variability in tail length was detected (P = 0.001), but head width (P = 0.114) and length (P = 0.069) did not differ significantly. Multivariate pattern analysis (PATN computer package) highlighted three sub-populations of spermatozoa objectively on the basis of tail length (10.0-22.0 microns, 22.1-73.0 microns and 73.1-130.0 microns). The Landrace-Meishan introgression boar possessed more spermatozoa (P < 0.0001) with tails 73.1-130 microns long. Subsequent analysis of morphology parameters in a pure-bred Meishan boar showed similar measurements for tail length (mean +/- SD; 66.36 +/- 24.70 microns) to the Landrace-Meishan introgression boar (mean +/- SD; 67.09 +/- 21.80 microns). Sperm subpopulations originate during spermatogenesis, when heterogeneous genotypic effects determine the structural features of spermatozoa. The findings of this study confirm that tail length differs between boars and that subpopulations of spermatozoa can be detected within a single ejaculate.

The development of a Children's Implant Profile.

The decision to provide a child with a cochlear implant is quite complex, as it must include consideration not only of the implant itself but also of the habilitative services necessary following the surgical procedure. To provide a systematic means of selecting hearing-impaired children for cochlear implants, a team at Children's Hearing Institute, Manhattan Eye, Ear and Throat Hospital, developed the Children's Implant Profile (ChIP). There is no one profile of a successful implant user--at least 11 factors appear to contribute to successful implantation. In the ChIP, each factor is evaluated on a three-point scale: (1) no concern, (2) mild-to-moderate concern, and (3) great concern. A profile showing "no concern" on all 11 factors denotes clear acceptability of the child as an implant candidate. A profile including several ratings in the "mild-to-moderate concern" category suggests a need for further study to determine if improvements could be made in projected outcomes before initiating surgical procedures. Finally, ratings of "great concern," especially on more than one factor, indicate a very limited probability of successful implant outcomes, at least at the time of evaluation. A case study is presented to demonstrate the relationship between the evaluated factors and to show how the profile is used to address and remedy areas of concern.

Inhibition of angiotensin III formation by thiol derivatives of acidic amino acids.

Angiotensin III is formed by removal of the N-terminal Asp residue of angiotensin II in a reaction catalyzed by glutamyl aminopeptidase (aminopeptidase A EC 3.4.11.7). Thiol derivatives of glutamate and aspartate in which the alpha-COOH group was replaced by -CH2SH were synthesized as inhibitors of glutamyl aminopeptidase. Glutamate thiol was a potent inhibitor of glutamyl aminopeptidase (Ki = 4 x 10(-7) M) but even more potently inhibited microsomal alanyl aminopeptidase (Ki = 2.5 x 10(-7) M). Aspartate thiol (beta-homocysteine) was a less potent but more selective inhibitor of glutamyl aminopeptidase (glutamyl aminopeptidase: Ki = 1.2 x 10(-6) M; microsomal alanyl aminopeptidase: Ki = 7.5 x 10(-6) M). Neither compound inhibited cytosolic leucyl aminopeptidase. Aspartate thiol blocked the conversion of angiotensin II to angiotensin III. These derivatives are more selective than amastatin and may be of value in studies probing the biological significance of angiotensin III.

Antitumor agents, 107. New cytotoxic 4-alkylamino analogues of 4'-demethyl-epipodophyllotoxin as inhibitors of human DNA topoisomerase II.

A series of analogues of etoposide, the C-4 amino- and alkylamino-substituted 4'-demethyl-epipodophyllotoxins, have been synthesized and studied for their activity to inhibit type II human DNA topoisomerase as well as their activity in causing cellular protein-linked DNA breakage. Substitution of the glycosidic moiety of 1 by a 2"-hydroxyethylamino or 2"-methoxyethylamino chain at the C-4 beta position resulted in potent inhibitors of the human DNA topoisomerase II. This inhibitory activity correlates reasonably well with their activity in causing protein-linked DNA breakage in KB cells. The in vitro cytotoxicity (KB) appears to have no correlation with the inhibitory activity of the human DNA topoisomerase II.

Antitumor agents. 100. Inhibition of human DNA topoisomerase II by cytotoxic ether and ester derivatives of podophyllotoxin and alpha-peltatin.

A principal mechanism of action of the clinical antitumor drugs etoposide (1) and teniposide (2) is the inhibition of catalytic activity of type II DNA topoisomerase and concurrent enzyme-mediated production of lethal DNA strand breaks. Substitution of the glycosidic moiety of 1 or 2 by ester and ethers, as well as the esterification and etherification of alpha-peltatin (4) including its glucosidic ethylidene and thenylidene cyclic acetals (25 and 26), has afforded compounds of much less activity than that of 1. The in vitro cytotoxicity (KB) appears to have no correlation with the inhibitory activity of the human DNA topoisomerase II.