Effects of pig follicular fluid on maturation of pig oocytes in vitro and on their subsequent fertilizing and developmental capacity in vitro. This study examines the effects of pig follicular fluid on the maturation of pig oocytes and on their subsequent fertilizing and developmental capacity in vitro. The addition of pig follicular fluid or its fractions obtained by ultrafiltration, gel filtration and ion-exchange chromatography to maturation medium significantly increased the rates of nuclear maturation, normal fertilization and normal cleavage of pig oocytes after fertilization in vitro: the rates of normal fertilization and cleavage were 2-4 times higher than those in the control medium. The efficacy of pig follicular fluid was lost after heating at 56 degrees C for 30 min, whereas no significant decrease in activity was observed after defatting. In addition, the effective component(s) was partially purified by ultrafiltration, gel filtration and ion-exchange chromatography: the activity was observed in the fraction (UF2; M(r) 10,000-20,000) obtained by ultrafiltration. Activity was found in the first fraction (G1) obtained by gel filtration of UF2. Among three fractions obtained by ion-exchange chromatography of G1, only the third fraction had the activity. The results indicate that pig follicular fluid contains an acidic substance(s) (M(r) 10,000-200,000) that promotes oocyte maturation.


Concentrations of immunoreactive inhibin in ovarian and peripheral venous plasma and follicular fluid of Booroola ewes that are homozygous carriers or non-carriers of the FecB gene. No gene-specific differences were found during either the luteal or follicular phases of the oestrous cycle in the venous secretion rates of ovaries or in concentrations of immunoreactive inhibin in peripheral plasma between Booroola ewes that were homozygous carriers (BB) or non-carriers (++) of the FecB gene. In three experiments in which concentrations of plasma inhibin and follicle-stimulating hormone (FSH) were compared, gene-specific differences were noted for FSH (P less than 0.05), but no significant correlations were noted between FSH and inhibin for either genotype. Granulosa cells and follicular fluid, but not theca interna, stroma or corpora lutea, were the major intra-ovarian sites of inhibin; no gene-specific differences were noted for inhibin concentrations in follicular fluid or in any of the intra-ovarian tissues. The mean concentrations of inhibin in follicular fluid remained constant irrespective of follicular diameter whereas the mean total contents of inhibin increased significantly with increasing diameter (P less than 0.05). Inhibin secretion rates were four times higher in ovaries with oestrogen-enriched follicles (i.e. greater than or equal to 50 ng oestradiol ml-1) than in ovaries with no such follicles (P less than 0.01). Moreover, inhibin concentrations were higher in follicular fluid of oestrogen-enriched follicles than in those with low oestrogen (i.e. less than 50 ng ml-1; P less than 0.05). Ovariectomy resulted in a significant reduction in concentrations of immunoreactive inhibin from plasma (P less than 0.01). The residual plasma inhibin in some Booroola ewes was not associated with genotype. It is concluded that, although antral follicles are a major source of inhibin in Booroola ewes, immunoreactive inhibin is not associated with the FecB gene and is not responsible for the gene-specific differences in concentrations of FSH in plasma.


The dynamic provision of different energy substrates improves development of one-cell random-bred mouse embryos in vitro. Preliminary observations showed that one-cell embryos from random-bred MF1 mice avoid cleavage arrest at the two-cell stage ('in vitro two-cell block') when cultured in modified M16 culture medium containing lactate and pyruvate but lacking glucose. The roles of lactate, pyruvate and glucose during preimplantation development of embryos from random-bred mice in vitro were therefore examined. When all three substrates were present continuously during culture, one-cell embryos arrested at the two- to four-cell stages. Improved development to the morula stage after 96 h in culture was obtained in media containing pyruvate alone, lactate and pyruvate, pyruvate and glucose, lactate pyruvate and glucose for the first 24 h, and medium containing lactate and pyruvate for the remaining 72 h. In a second experiment, embryos were cultured in medium containing pyruvate alone, lactate and pyruvate or pyruvate and glucose for the first 24 h, and lactate plus pyruvate medium for the second 24 h. Subsequent transfer to medium containing lactate, pyruvate and glucose supported the morula to blastocyst transition. These results show that developmental arrest in vitro can be overcome by changing the combination of energy substrates at different stages of preimplantation development.


Histology of ovaries of female rabbits immunized with deglycosylated zona pellucida macromolecules of pigs. Female rabbits (n = 36, 6 per group) were immunized with: (i) solubilized isolated porcine zona pellucida (SIZP), which contains ZP1, 82 kDa; ZP3 alpha, 55 kDa; and ZP3 beta, 55 kDa; (ii) a purified preparation of ZP3 alpha and ZP3 beta (ZP3); (iii) purified endo-beta-galactosidase digested glycoproteins ZP3 alpha-(EBGD) and (iv) ZP3 beta-(EBGD) (each about 30% deglycosylated); (v) chemically deglycosylated core proteins ZP3 alpha-(DG) and (vi) ZP3 beta-DG (each greater than 92% deglycosylated). Rabbits injected with saline (n = 6) or Freund's adjuvant (n = 6) served as controls. Rabbits were bled weekly to monitor titres. Every six weeks two animals from each group (n = 16) were selected for unilateral oophorectomy followed by histological examination. Sections were scored for numbers of primary, secondary and tertiary follicles. Anti-ZP3 titres developed in all treatment groups and correlated with carbohydrate content (peak per cent [125I]-labelled ZP3 binding by radioimmunoassay: SIZP 71.9 +/- 1.2, ZP3 70.0 +/- 2.5, ZP3 alpha-EBGD 60.9 +/- 5.3, ZP3 beta-EBGD 56.4 +/- 5.0, ZP3 alpha-DG 56.4 +/- 4.0, ZP3 beta-DG 53.5 +/- 4.3) (means +/- SEM). Animals immunized with SIZP, ZP3 and ZP3 beta-EBGD showed a statistically significant reduction in the number of primary, secondary and tertiary follicles compared with controls (P less than 0.01, MANOVA), whereas animals immunized with ZP3 alpha-EBGD, ZP3 alpha-DG and ZP3 beta-DG did not (P greater than 0.05, MANOVA). These results demonstrate that immunization with purified ZP3 alpha macromolecules (ZP3 alpha-EBGD, ZP3 alpha-DG) or ZP3 beta-DG does not produce histopathological changes in ovaries. Such deglycosylated ZP macromolecules represent potential target antigens for immunocontraceptive development.


Embryo-initiated oviductal transport in mares. The hypothesis that equine embryos initiate oviductal transport in mares was tested by placing day 6 uterine embryos in the oviducts of day 2 (n = 10) or day 5 (n = 10) recipient mares and attempting to collect the embryos from the uterus 48 h later. To determine whether the surgical transfer procedure initiated oviductal transport, medium alone was placed in the oviducts of day 2 (n = 10) inseminated mares (sham transfer), and uterine embryo collections were attempted 48 h later. Embryos were transported through the oviduct of day 2 recipients by day 4 (instead of day 5 to 6) in six of ten mares, which was not significantly less (P greater than 0.1) than in day 5 recipients (9 of 10). Oviductal transport was not primarily initiated by the surgical transfer procedure, since oviductal transport occurred in only one sham transfer. There was no significant difference (P greater than 0.1) in the diameter of embryos placed in the oviducts of day 2 and day 5 recipient mares (180 +/- 13.8 versus 187 +/- 11.3 microns, respectively). However, embryos collected from the uterus were significantly smaller (P less than 0.05) in day 2 than in day 5 recipients (375 +/- 85.4 versus 659 +/- 43.6 microns, respectively). One uterine embryo had shed its zona pellucida before being placed in, and transported through, the oviduct of the recipient mare.


Localized increases in ovarian vascular permeability and leucocyte accumulation after induced ovulation in rabbits. Colloidal carbon was injected i.v. in mature virgin rabbits at different times after induction of ovulation by human chorionic gonadotrophin (hCG, 100 iu) or mating. Before induction of ovulation, slight carbon leakage was observed in the inner vascular ring of the theca interna of antral follicles, but blood vessels in the other ovarian compartments were unstained. Between 4 and 10.5 h after hCG-treatment or mating, vascular leakage was most marked in the blood vessels of the interstitial gland and in the theca interna of antral follicles. Just before ovulation, carbon particles were observed between granulosa cells and some carbon was seeping into the follicular fluid of preruptured follicles. Vascular leakage was also observed over the follicle dome before rupture as well as at the dorsomedial junction between the mesovarium and the ovary. The blood vessels stained with carbon were 7-70 microns diameter, representing capillaries and postcapillary venules. About 6 h after hCG injection, an increased number of polymorphonuclear leucocytes migrated from the vessels of these ovarian compartments into the surrounding interstitial tissue. The number of leucocytes seen in the follicular wall and ovarian medulla increased markedly towards ovulation. During early corpus luteum formation, the number of leucocytes decreased markedly. The localized vascular changes seen after mating and hCG stimulation were similar to an inflammatory reaction and could form the basis for the formation of peritoneal exudate after ovulation in rabbits and periovulatory ascitic accumulation seen in the peritoneal cavity of women during the menstrual cycle.


Increase in uterine peroxidase activity in the rat uterus during oestrogen hyperaemia. The administration of oestrogen results in increased arterial blood flow in all mammalian species studied to date, but its mechanism of action has not been elucidated. Because an interval of 30-60 min is observed between oestrogen injection and uterine hyperaemia, it has been suggested that a vasoactive intermediate is involved and recent evidence suggests that catechol oestrogens are the vasoactive oestrogen intermediates. Uterine peroxidase catalyses the conversion of oestrogens to their catechol forms and thus may play an important role in oestrogen-induced uterine hyperaemia. The present studies evaluated the time course and dose-response effects of oestrogen on uterine peroxidase activity and related these to changes in uterine blood volume, an index of uterine hyperaemia in immature rats. These data demonstrated that the minimal effective hyperaemic dose of oestradiol also increased (P less than 0.05) uterine peroxidase activity. The oestradiol-induced increase in uterine peroxidase activity preceded significant increases in uterine blood volume (1 h versus 2 h, respectively). These data are consistent with a role for peroxidase-mediated conversion of oestradiol to catechol oestradiol in facilitating uterine hyperaemia in rats.


Localization of type I interferon in murine trophoblast and decidua during decidual formation. Mouse trophoblast and decidua were examined by means of immunohistochemistry to define the localization of type I interferon. The decidua were stained for type I interferon at the time of implantation. The strong reaction was first observed in the primary decidual zone on day 5 and subsequently in the secondary decidual zone on day 6. After day 10, the decidua basalis and decidua capsularis showed a strong reaction. At the one-cell stage, embryos were weakly labelled, but a positive reaction was recognized in compacted morulae. Blastocysts on days 3 and 4 were positive in trophoblast and inner cell mass and a strong reaction was observed in the primitive endoderm on day 4. The visceral endoderm on day 5 and the trophoblast on day 6 were positive. After day 10, the trophoblast giant cells, labyrinth, visceral yolk sac and fetal blood cells gave a positive reaction. This study is the first demonstration of type I interferon localization in situ in mouse trophoblast and decidua during decidual formation.


Influence of a gonadotrophin-releasing hormone agonist and gonadotrophins on morphometric characteristics of the population of small ovarian follicles in cynomolgus monkeys (Macaca fascicularis). Small follicles, less than or equal to 100 microns, in monkey ovaries were divided into four types based on the morphological characteristics of the granulosa cells that surrounded the oocyte: primordial, intermediary, primary and secondary follicles. The proportion of primordial follicles positively correlated, whereas those of intermediary, primary and secondary follicles negatively correlated, with the total number of follicles less than or equal to 100 microns. There was no relationship between the population of nongrowing follicles (primordial and intermediary) and that of early-growing follicles (primary and secondary). Administration of exogenous gonadotrophins did not induce significant changes in the population of small follicles, whereas there was a significant increase in the number of intermediary follicles when gonadotrophins were associated with a gonadotrophin-releasing hormone agonist, buserelin. Buserelin can therefore partly inhibit the initiation of ovarian follicular growth in monkeys.


Effect of human chorionic gonadotrophin on dominant follicles in cows: formation of accessory corpora lutea, progesterone production and pregnancy rates. Thirty-four lactating Holstein cows were randomly assigned to four groups for treatment with human chorionic gonadotrophin (hCG, 1000 iu) at insemination day 0 (n = 8) or 7 (n = 9) or 14 days (n = 9) after insemination or with no hCG treatment (control, n = 8). Ultrasound imaging of the ovaries and plasma progesterone measurements were carried out to determine follicular dynamics and corpus luteum growth and function. Rates of formation of accessory corpora lutea were higher among cows treated on days 0 (three cows), 7 (seven cows) or 14 (four cows) than in the controls (one cow). Total corpus luteum diameter was greater (P less than 0.01) among hCG-treated cows than in controls 7-42 days after insemination. Concentrations of progesterone in plasma were significantly (P less than 0.05) higher in cows treated with hCG on days 7 or 14 than in those treated on day 0 or in controls, at days 18, 35 or 42 after insemination. Seven of the cows treated on day 7 became pregnant, whereas four, four and three cows treated on days 0 or 14 and control cows became pregnant, respectively. The results suggest that hCG treatment at 7 days after insemination could be used to produce accessory corpora lutea, raise plasma progesterone concentration and hence reduce the incidence of early embryonic mortality in cattle.


Developmentally related changes in the metabolism of glucose and glutamine by cattle embryos produced and co-cultured in vitro. The metabolism of radiolabelled glucose and glutamine was measured in individual cattle embryos produced by in vitro maturation and fertilization of oocytes, and culture with bovine oviductal epithelial cells. Metabolism of glucose through the pentose-phosphate pathway increased almost 15 times and the total metabolism of glucose 30 times, during development from the two-cell to the expanded blastocyst stage. The first marked increase in glucose metabolism did not occur until between the eight- and 16-cell stages, the time of activation of the embryonic genome. Conversely, the metabolism of glutamine was high in two- and four-cell embryos and then decreased to reach a minimum at the compacted morula to blastocyst stage, possibly because of degradation of maternally derived enzymes. Blastocyst expansion was accompanied by significant increases in the metabolism of glucose and glutamine, presumably reflecting the increased energy demands of Na(+)-K+ ATPase necessary for formation and maintenance of the blastocoel.


In vitro maturation, fertilization and development of follicular oocytes from buffalo (Bubalus bubalis). Cumulus-oocyte complexes, 5596, were cultured for 24 h in either TCM-199 or Ham's F-10 with or without gonadotrophins and supplemented with either 20% buffalo oestrous serum (BES) or fetal calf serum (FCS). The maturation rates of oocytes cultured in TCM-199 or Ham's F-10 medium supplemented with 20% BES were 47.4 +/- 17.8 and 44.8 +/- 25.6, respectively. Addition of luteinizing hormone (LH) (5 micrograms ml-1) significantly improved the maturation rate in the Ham's F-10 medium supplemented with 20% BES (76.8 +/- 18.3), but follicle-stimulating hormone (FSH) (0.5 micrograms ml-1) and oestradiol (1 microgram ml-1) failed to synergize with LH (71.7 +/- 19.5). In the TCM-199 system, LH failed to enhance the maturation rate but the addition of FSH and oestradiol significantly enhanced the proportion of mature oocytes (42.7 +/- 1.4 and 81.7 +/- 14.5, respectively; P less than 0.05). Frozen-thawed spermatozoa prepared in Bracket and Oliphant (BO) medium and treated with 5 mmol caffeine 1(-1) + 10 micrograms heparin showed a higher fertilization rate (29.8%) than those treated in Hepes-Talp and treated with 10 micrograms heparin ml-1 (19.6%). Fertilization rate was significantly improved when fresh ejaculated spermatozoa treated with 5 mmol caffeine 1-1 and 10 micrograms heparin in BO medium (50%) was used. Rate of cleavage and development were also higher when in vitro fertilization was carried out with fresh ejaculated spermatozoa treated with caffeine and heparin (34.1 and 36.8%, respectively) than with frozen-thawed spermatozoa (27.0 and 22.0%, respectively). Development rate was enhanced when fertilized ova were cultured in ligated rabbit oviduct (28.0%) than when co-cultured on oviductal cell monolayers (8.2%). The results indicate that oocytes cultured in medium supplemented with BES and gonadotrophins reveal high rates of maturation and development to the blastocyst stage after fertilization with fresh ejaculated spermatozoa.


Interbirth interval and duration of pregnancy in hares. The possibility of inducing superfetation in hares by artificial insemination (AI) was investigated. AI performed on various days during the second half of gestation did not result in new pregnancies, but all ongoing pregnancies were terminated 1-4 days after AI. We suggest that copulation during the last week of pregnancy, common among captive hares, may have a similar effect to that of AI in terminating pregnancies and inducing early deliveries. Intervals between successive deliveries that are shorter than the normal duration of gestation do not necessarily indicate superfetation and this phenomenon may be rare among hares in captivity as well as in the wild.


Effects of removal of chicks from hens on concentrations of prolactin, luteinizing hormone and oestradiol in plasma of brooding Gifujidori hens. Gifujidori hens were allowed to repeat a breeding cycle in one season. In the first breeding cycle the duration of the brooding (raising chicks) stage was limited to 3 weeks, whereas in the second breeding cycle it was limited to 1 week by removing all chicks from mother hens. In the first breeding cycle, plasma prolactin (PRL) was high during the incubation period, but rapidly decreased on the day of hatching and reached minimum values about 1 week after hatching. In contrast, plasma luteinizing hormone (LH) concentrations were low during the incubation period, but after hatching they gradually increased and reached peak values immediately after removal of chicks. Concentrations of oestradiol in plasma were low in the incubation and brooding stages but increased significantly immediately after removal of chicks. In the second breeding cycle, changes in PRL and LH concentrations were similar to those observed in the first breeding cycle except that even greater increases in plasma LH and oestradiol concentrations were observed one week after hatching when the chicks were removed. These results suggest that coexistence of newly hatched chicks may suppress LH secretion from the pituitary of the hen in the natural breeding cycle.


Endothelin-like immunoreactivity in human endometrium. Endothelin-like immunoreactivity (ET-IR) was detected immunocytochemically in glandular epithelium and vascular endothelium of human endometrium and myometrium. Primary antibody was raised in rabbits against the carboxy-terminal heptapeptide of endothelin 1 (ET-1), ET-1(15-21), and compared with antibodies raised against the cyclized amino-terminal, ET-1(2-13), and commercially obtained antibodies against the whole ET-1 or ET-3 molecule. Binding was visualized using the peroxidase technique in sections counter-stained with haemalum. Staining was seen in each of 15 sections from eight women in the proliferative (five) or secretory (three) phase of the cycle. Intense staining was present in the cytoplasm of endometrial glands and vascular endothelium, and was greatest at the endometrial-myometrial junction. The pattern of staining was similar with all primary antibodies tested. The demonstration of ET-IR in endometrium suggests that the endothelins may play a role in control of the uterine vascular bed.


Retardation of pubertal development by prenatal long days in goat kids born in autumn. Goat kids born in spring attain sexual maturity during the first autumn after birth in temperate regions, at about 30 weeks of age. This study observed sexual development in autumn-born kids and the influence of late-summer, prenatal light treatment on onset of puberty. The breeding season of 14 female British Saanen dairy goats was artificially advanced by 4 months, using a treatment of long days during the winter followed by melatonin treatment in spring. Five goats were treated with a photoperiod of 20 h light:4 h dark (lights on 04.00 h) for 62.1 +/- 1.4 days (mean +/- SEM, n = 5) prepartum (14 August to 15 October). The remaining nine goats were kept under a natural photoperiod: 20 kids from these mothers were followed, five males and five females from each group. Testicular development was assessed by means of weekly measurement of scrotal circumference. Blood samples were taken once a week from all kids from 4 weeks of age for 5 months. Plasma was assayed for progesterone in females and testosterone in males. Autumn-born female kids initiated oestrous cyclicity in January, at a mean age of 12.8 +/- 0.8 weeks. Puberty onset was significantly delayed (P less than 0.03, unpaired Student's t test) in females exposed to 20 h light:4 h dark in utero and occurred at a mean age of 16.5 +/- 1.4 weeks. Testicular development was significantly delayed and plasma testosterone concentrations were lower in autumn-born male kids that experienced 20 h light:4 h dark in utero than in kids from mothers in a natural photoperiod.(ABSTRACT TRUNCATED AT 250 WORDS)


Hereditary haemorrhagic telangiectasia: a clinical analysis. Data from 98 patients with hereditary haemorrhagic telangiectasia (HHT) are presented. All were symptomatic by 40 years of age and 62% by 16 years. Nose bleeding was the first symptom of disease in 90% of cases with mucocutaneous telangiectases appearing 5 to 20 years later. Complications of HHT are discussed and an age of onset curve given.


Genetic transmission of colorectal cancer: exploratory data analysis from a population based registry. Classical segregation analysis was conducted on 605 families of probands with colorectal carcinoma ascertained through the Cancer Registry of the Province of Modena in Italy. The families were classified as 28 suspected hereditary non-polyposis colorectal cancer (HNPCC) syndromes and 577 presumed non-HNPCC. In 11 of these, both parents had colorectal carcinoma, in 130 one parent was affected, and in 436 both parents were normal. In the suspected HNPCC families, segregation was compatible with dominant transmission of susceptibility to carcinoma. In families with one parent affected, the segregation frequency was almost exactly equal to the frequency of segregation in families where both parents were normal. The model of dominant transmission of susceptibility through a major gene with greatly reduced penetrance in heterozygotes fitted the data acceptably.


Comparison of adult height between patients with XX and XY gonadal dysgenesis: support for a Y specific growth gene(s). Adult height was compared between published cases of patients with XX gonadal dysgenesis (XXGD) and those with XY gonadal dysgenesis (XYGD). The mean adult height of XYGD patients (171.0 cm (SD 7.8), n = 27) was significantly greater than that of XXGD patients (164.4 cm (7.7), n = 27) (p less than 0.01). This finding supports the existence of a Y specific growth gene(s) which promotes statural growth independently of the effects of gonadal sex steroids.


Minute Y chromosome derived marker in a child with gonadoblastoma: cytogenetic and DNA studies. A 12 year old girl referred for chromosome analysis because of short stature was found to have karyotype mos 45,X/46,X,+mar. The marker chromosome was observed in 58% of her blood lymphocytes. It was a small, pale staining, spherical fragment with GTL banding and showed faint differentiation along its length with CBG banding. DNA analysis using Y specific probes showed the absence of the testicular determining region and the presence of some short arm and centromeric Y chromosomal material. In situ hybridisation confirmed that the Y chromosomal material was associated with the marker chromosome. At laparotomy the patient was found to have streak gonads. Gonadectomy was subsequently performed and histological examination showed dysgenetic gonads with a dysgerminoma arising from a gonadoblastoma in the left gonad. This case shows that even very small Y derived marker chromosomes with pericentric material can predispose the phenotypic female to gonadal neoplasia.


Racial distribution of Duchenne muscular dystrophy in the West Midlands region of Britain. In the West Midlands region of Britain, Duchenne muscular dystrophy (DMD) is twice as common as expected in Indians, and is less common than expected in Pakistanis. Although the numbers are small, they cannot be explained by any bias of ascertainment and are considered to be real. One possible mechanism for the high frequency of DMD in Indians is the presence of repetitive elements in the wild type gene which predispose to mutations.


Investigation of a female manifesting Becker muscular dystrophy. Females manifesting Becker muscular dystrophy (BMD) are even more rarely observed than for the allelic condition Duchenne muscular dystrophy. The male proband has typical BMD with greatly raised CK activity and a myopathic muscle biopsy. His mother experienced walking difficulties from 35 years of age and has a myopathy with marked calf hypertrophy, a raised CK, and a myopathic muscle biopsy. Dystrophin analysis was undertaken on both the proband and his mother. Immunoblotting showed a protein of normal size but of reduced abundance in both. Immunocytochemical analysis in the proband indicated that the majority of the fibres showed weak dystrophin labelling and in his mother both dystrophin positive and dystrophin negative fibres were present. Non-random X inactivation at locus DXS255, was observed in DNA isolated from peripheral lymphocytes of the mother. Neither extended multiplex PCR performed on DNA from the proband nor analysis of lymphocyte derived mRNA showed a structural alteration in the dystrophin gene suggesting that an unusual mutation was responsible for BMD in this family.


Congenital hydrocephalus secondary to Walker-Warburg syndrome identified on the Manitoba Neonatal Screening Programme for Duchenne muscular dystrophy. This report describes our first experience with a clinically important true false positive neonatal screening test for Duchenne muscular dystrophy (DMD). Neonatal screening for DMD began as a pilot programme in Manitoba on 1 January 1986 by analysis of creatine kinase (CK) activity in dried filter paper blood spots. To date, all except two males with positive initial and follow up neonatal CK screening tests were subsequently diagnosed as having DMD. Of these two, one was a newborn male with congenital hydrocephalus whose positive DMD screening test led to the identification of an associated congenital myopathy and confirmation of the diagnosis of Walker-Warburg syndrome.


Simultaneous partial monosomy 10p and trisomy 5q in a case of hypoparathyroidism. We report a case of monosomy for the distal region of the short arm of chromosome 10 (p13----ter) associated with trisomy for the terminal region of the long arm of chromosome 5 (q35.2----ter) that had originated from adjacent 1 segregation of a maternal reciprocal balanced translocation (5;10)(q35.2;p13). We review the clinical findings of previously reported cases of both partial monosomy for 10p and of partial trisomy for 5q, but to our knowledge there are no previous reports of the effects of these two chromosome anomalies together. Clinically our patient showed features typical of partial monosomy for 10p (including hypothyroidism) rather than partial trisomy 5q.


Upper limb defect associated with developmental delay, unilateral poorly developed antihelix, hearing deficit, and bilateral choroid coloboma: a new syndrome. Two sibs are reported with upper limb defect, developmental delay, central hearing loss, unilateral poorly developed antihelix, and bilateral choroid coloboma. The inheritance is probably autosomal recessive.


Progressive vitiligo, mental retardation, facial dysmorphism, and urethral duplication without chromosomal breakage or immunodeficiency. A boy, born to first cousin parents of Algerian origin, first presented at the age of 9 years with growth failure, mental retardation, and dysmorphic facies. Progressive vitiligo developed from the age of 12 and distal duplication of the urethra was later recognised. The basis of this syndrome remains to be determined; autoimmune disease, chromosomal breakage syndromes, and other neurocutaneous syndromes have been excluded.


Comparison of a blood-free medium and a filtration technique for the isolation of Campylobacter spp. from diarrhoeal stools of hospitalised patients in central Australia. Single specimens of diarrhoeal stool from 676 patients, mostly aboriginals aged less than 5 years, admitted to Alice Springs Hospital, central Australia, for diarrhoea between Sept. 1988 and Feb. 1989, were examined for Campylobacter spp. by culture on a blood-free medium with selective supplement (BFM; Oxoid) and blood agar overlaid with a membrane filter (FM). Campylobacter spp. were isolated on either BFM or FM or both from 225 patients. Campylobacter spp. were isolated on BFM alone from 75 patients and on FM alone from 213 patients (p less than 0.001; chi 2 test). Most campylobacters isolated on BFM were C. jejuni. All C. jejuni subsp. doylei, all "C. upsaliensis" except one, all C. laridis, C. fetus subsp. fetus and several uncharacterised Campylobacter isolates were isolated on FM only. C. jejuni was isolated on BFM but not FM from several patients, and vice versa. Serotyping of C. jejuni and C. coli isolated from both media showed the serotypes recovered from the two media to be different in some patients. In some patients concurrent infection with several species or serotypes (up to five) of Campylobacter, or both, was shown for the first time by the use of FM. We conclude that the use in combination of a selective medium and a non-selective medium with a filtration technique are better than either medium alone for the isolation of Campylobacter spp.


Carotenoid pigment levels in Staphylococcus aureus and sensitivity to oleic acid. Staphylococcus aureus mutants lacking pigment, or expressing only low levels of pigment, were more sensitive to oleic acid than were the parent strain and mutants making more pigment than the parent. One class of mutants (colour index 5), although possessing significant levels of pigment, were nevertheless quite sensitive to oleic acid. This suggested that only certain carotenoids in the biosynthetic pathway were capable of imparting resistance to fatty acids. The phenotypic expression of pigment also affected the sensitivity of a strain to oleic acid. The parent S. aureus strain 18Z, when grown to express its maximal pigment potential, was more resistant to oleic acid than when it was grown to express minimal levels of pigment.


The typing of Staphylococcus epidermidis by a lectin-binding assay. A new typing method for Staphylococcus epidermidis was developed. Four biotinylated lectins--wheat germ agglutinin (WGA), soy bean agglutinin (SBA), lentil agglutinin (LCA) and Concanavalin A (ConA)--were added to immobilised whole cells of coagulase-negative staphylococci (CNS) in microtitration plates. The amount of bound lectin was measured by peroxidase-conjugated avidin followed by a peroxidase reaction. The method was compared to antibiotic-resistance analysis, phage typing, plasmid DNA profiles and slime production. A total of 113 isolates of CNS from 21 patients was investigated and 71 strains of CNS, including 64 strains of S. epidermidis, were detected if all typing methods were taken into consideration. If only one typing method was used the highest discriminatory power among the S. epidermidis isolates was obtained with the lectin-binding assay which allowed 49 different strains to be detected. If the lectin-binding assay was combined with plasmid-profile analysis, all 64 different strains could be identified. The typability of lectin-binding assay was 96.9% among the S. epidermidis isolates and 25 different lectin-binding patterns were established among the 64 strains. The highest number of strains belonging to one lectin-binding pattern was 13 (20.3%). The assay was reproducible, easy to perform, relatively inexpensive and therefore applicable to large scale typing of S. epidermidis.


Purification and characterisation of elastase from Staphylococcus epidermidis. An elastase of Staphylococcus epidermidis was purified by ion exchange chromatography on CM-Sepharose and characterised. Its M(r) is c. 21 kDa, its optimal temperature for activity is 42 degrees C and the pH optimum is 6.8. The enzyme is activated by cysteine and other SH-donators and inhibited by L-trans-epoxy-succinylleucylamido-(4-guanidino)butane (E64), an inhibitor of cysteine proteases, but not by 3,4-dichloroisocoumarin (3,4-DCI), an inhibitor of serine proteases. This finding suggests that the elastase of S. epidermidis is a cysteine protease. Because S. epidermidis elastase degrades human sIgA, IgM, serum albumin, fibrinogen, and fibronectin, this enzyme may be regarded as a virulence factor.


Correlation between cytotoxin production and sporulation in Clostridium difficile. Correlation between cytotoxin production and sporulation was demonstrated when a Clostridium difficile culture was inoculated into fresh broth to give an initial count of less than 10 vegetative cells/ml with no spores. Under these conditions, cytotoxin was produced and released during sporulation. Addition of a sporulation inhibitor (acridine orange, 30 mg/L), resulted in a marked decrease in both sporulation and cytotoxin production, despite there being no change in the number of vegetative cells in the culture. These results indicate that sporulation might be closely related to cytotoxin production.


Restricted distribution of Streptococcus milleri carbohydrate type antigens amongst other viridans streptococci. The distribution of oral Streptococcus milleri carbohydrate type antigens in other viridans streptococcus species was examined. Rantz-Randall extracts of cells of the test strains grown in broth containing glucose were allowed to react with typing or grouping antisera for S. milleri serotypes a-k, or Lancefield groups A-G and K. Of 93 strains comprising more than 12 streptococcal species that included S. mutans and S. sanguis complexes, only 15 S. salivarius strains and one S. mitis strain were immunologically related to S. milleri serotype f. Unlike S. milleri strains, S. salivarius type f strains belonged to Lancefield group K, whereas the S. mitis strain was closely related to S. milleri serotype f but did not react with any of the Lancefield grouping antisera tested. Results suggest that oral S. milleri strains can be distinguished serologically from other oral viridans streptococci and that the typing antisera used in our researches might differentiate S. milleri isolates from the mouth from those associated with systemic purulent infections.


Monoclonal antibodies to a 16-kDa antigen of Serpulina (Treponema) hyodysenteriae. Monoclonal antibodies (MAbs) were produced to an outer-envelope preparation from Serpulina (Treponema) hyodysenteriae, the aetiological agent of swine dysentery. Three MAbs (isotype IgG1) were obtained. All three recognised a 16-kDa antigen that was common to most strains of S. hyodysenteriae of different serotypes but was absent from nonpathogenic, porcine intestinal spirochaetes. Immunofluorescence and immunogold labelling studies showed that the 16-kDa antigen was exposed on the surface of intact spirochaetes. The MAbs agglutinated freshly grown cultures of spirochaetes and inhibited growth of S. hyodysenteriae strains in vitro.


In-vitro susceptibility of Giardia lamblia to albendazole, mebendazole and other chemotherapeutic agents. The susceptibility of a strain of Giardia lamblia to benzimidazole carbamates, 5-nitroimidazoles, nitrofurans and other drugs was studied in vitro. Albendazole was the most active compound, with a 50% inhibitory concentration (IC50) of 0.01 mg/L and a minimal lethal concentration (MLC) of less than 0.04 mg/L; the IC50 of mebendazole was 0.06 mg/L and the MLC less than 0.5 mg/L. Among the 5-nitroimidazoles tested, ornidazole was the most effective (IC50 0.12 mg/L); tinidazole, metronidazole, secnidazole and hemezole were less active. Nifuroxazide, etofamide and nalidixic acid exhibited modest anti-giardial activity; quinfamide did not inhibit the growth of the parasite at a concentration of 200 mg/L. Albendazole and mebendazole are promising candidates for clinical use and should be further evaluated.


Chromatin structure of Schizosaccharomyces pombe. A nucleosome repeat length that is shorter than the chromatosomal DNA length. We have used new methods for chromatin isolation, together with conventional methods for measuring the nucleosome repeat length, to determine the repeat length of Schizosaccharomyces pombe chromatin. We obtain a result of 156(+/- 2) bp. Equivalent results are obtained using a psoralen crosslinking method for measuring the repeat length in viable spheroplasts. That result, together with other control experiments, rules out many possible artifacts. The measured value of 156(+/- 2) bp is smaller than the length of DNA found in the chromatosome. Thus, the chromatosome cannot be the fundamental unit of chromatin structure in all eukaryotes. The crossed linker model of chromatin higher order structure is incompatible with a nucleosome repeat length of 156 bp, and thus cannot apply to all eukaryotes. The solenoid model of higher order structure is compatible with this repeat length only if the solenoid is right-handed. We note two other properties of this chromatin. (1) Early in digestion, the DNA length of mononucleosomes from S. pombe and Aspergillus nidulans exceeds the nucleosome repeat length. (2) Many methods for isolating chromatin from S. pombe yield an apparent nucleosome repeat length of less than or equal to 140 bp; this result is found to be an artifactual consequence of nucleosome sliding.


Effect of sequence mutations on the higher order structure of the yeast 5 S rRNA. Mutant yeast ribosomal 5 S RNAs were probed by enzymatic cleavage and chemical reactivity to define further the higher order structure. Mutations that destabilized helix IV resulted in an altered tertiary structure in which a reduced reactivity to ethylnitrosourea at U90 and G91 could be correlated with greater enzymatic and Fe(II)-EDTA cleavages in helices II and V. The results provide direct evidence for, and a further definition of, a structural juxtaposition between helix II and the end of helix IV and indicate that, in contrast to earlier suggestions, the remaining tertiary structure is sufficiently stable to prevent "pseudoknot-like" interactions between helices III and IV. The data are fully consistent with the "lollipop" model of the tertiary structure.


Sporulation regulatory protein GerE from Bacillus subtilis binds to and can activate or repress transcription from promoters for mother-cell-specific genes. The mother-cell line of gene expression during sporulation in Bacillus subtilis is a hierarchical cascade consisting of at least four temporally controlled gene sets, the first three of which each contain a regulatory gene for the next gene set in the pathway. gerE, a member of the penultimate gene set, is a regulatory gene whose products is required for the transcriptional activation of genes (coat protein genes cotB and cotC) in the last gene set. The gerE product also influences the expression of other members of the penultimate gene set (coat protein genes cotA and cotD appear to be repressed and activated, respectively). We now report that the purified product of gerE (GerE) is a DNA-binding protein that adheres to the promoters for cotB and cotC. We also show that GerE stimulates cotB and cotC transcription in vitro by RNA polymerase containing the mother-cell sigma factor sigma K. These findings support the view that GerE is a positively acting, regulatory protein whose appearance at a late stage of development directly activates the transcription of genes in the last known temporal class of mother-cell-expressed genes. In addition, GerE stimulates cotD transcription and inhibits cotA transcription in vitro by sigma K RNA polymerase, as expected from in vivo studies, and, unexpectedly, profoundly inhibits in vitro transcription of the gene (sigK) that encodes sigma K. The effects of GerE on cotD and sigK transcription are just the opposite of the effects exerted by the earlier-appearing, mother-cell regulatory protein spoIIID, suggesting that the ordered appearance of first SpoIIID, then GerE, ensures proper flow of the regulatory cascade controlling gene expression in the mother cell.


Sequences of 20 subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Application of a novel strategy for sequencing proteins using the polymerase chain reaction. NADH:ubiquinone oxidoreductase, the first enzyme in the respiratory electron transport chain of mitochondria, is a membrane-bound multi-subunit assembly, and the bovine heart enzyme is now known to contain about 40 different polypeptides. Seven of them are encoded in the mitochondrial DNA; the remainder are the products of nuclear genes and are imported into the organelle. The primary structures of 12 of the nuclear coded subunits have been described and those of a further 20 are described here. The subunits have been sequenced by following a strategy based on the polymerase chain reaction. This strategy has been tailored from existing methods with the twofold aim of avoiding the use of cDNA libraries, and of obtaining a cDNA sequence rapidly with minimal knowledge of protein sequence, such as can be determined in a single N-terminal sequence experiment on a polypeptide spot on a two-dimensional gel. The utility and speed of this strategy have been demonstrated by sequencing cDNAs encoding 32 nuclear-coded-membrane associated proteins found in bovine heart mitochondria, and the procedures employed are illustrated with reference to the cDNA sequence of the 20 subunits of NADH:ubiquinone oxidoreductase that are presented. Extensive use has also been made of electrospray mass spectrometry to measure molecular masses of the purified subunits. This has corroborated the protein sequences of subunits with unmodified N terminals, and their measured molecular masses agree closely with those calculated from the protein sequences. Nine of the subunits, B8, B9, B12, B13, B14, B15, B17, B18 and B22 have modified alpha-amino groups. The measured molecular masses of subunits B8, B13, B14 and B17 are consistent with the post-translational removal of the initiator methionine and N-acetylation of the adjacent amino acid. The initiator methionine of subunit B18 has been removed and the N-terminal glycine modified by myristoylation. Subunits B9 and B12 appear to have N-terminal and other modifications of a hitherto unknown nature. The sequences of the subunits of bovine complex I provide important clues about the location of iron-sulphur clusters and substrate and cofactor binding sites, and give valuable information about the topology of the complex. No function has been ascribed to many of the subunits, but some of the sequences indicate the presence of hitherto unsuspected biochemical functions. Most notably the identification of an acyl carrier protein in both the bovine and Neurospora crassa complexes provides evidence that part of the complex may play a role in fatty acid biosynthesis in the organelle, possibly in the formation of cardiolipin.(ABSTRACT TRUNCATED AT 400 WORDS)


New triple-helical model for the shaft of the adenovirus fibre. The adenovirus fibre is a trimeric protein with a globular head on a long thin shaft that projects from the twelve fivefold vertices of the virion. The shaft region of the fibre primary sequence has a unique pseudo-repeating motif of 15 residues. Using constraints derived from sequence analysis, the trimeric nature of the fibre, the experimental determination of the shaft length and general knowledge about protein structure, an atomic model of the fibre shaft has been constructed by computer modelling techniques. In the final model the three monomers form a left-handed triple-helical structure with threefold symmetry and with successive 15-residue repeats on the same chain related by an axial rise of 13.1 A and a left-handed azimuthal rotation of close to 300 degrees. Three threefold related beta-sheets with short strands are formed by inter-monomer main-chain hydrogen bonds giving rise to superhelical ribbons covering the surface of the shaft. The model satisfies criteria of extensive hydrogen bonding, reasonable backbone torsion angles, burial of most hydrophobic residues and good packing of the hydrophobic core. Furthermore, the model is consistent with the observed shaft length of about 290 A and its calculated X-ray fibre diffraction patterns shows the characteristic features found in the diffraction pattern of crystals of fibre, notably layer lines with a spacing of about 1/26 A-1 and strong meridional intensity at 1/4.4 A-1.


Refined 2.3 A X-ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-based thrombin inhibitors NAPAP, 4-TAPAP and MQPA. A starting point for improving antithrombotics. Well-diffracting crystals of bovine epsilon-thrombin in complex with several "non-peptidic" benzamidine and arginine-based thrombin inhibitors have been obtained by co-crystallization. The 2.3 A crystal structures of three complexes formed either with NAPAP, 4-TAPAP, or MQPA, were solved by Patterson search methods and refined to crystallographic R-values of 0.167 to 0.178. The active-site environment of thrombin is only slightly affected by binding of the different inhibitors; in particular, the exposed "60-insertion loop" essentially maintains its typical projecting structure. The D-stereoisomer of NAPAP and the L-stereoisomer of MQPA bind to thrombin with very similar conformations, as previously inferred from their binding to bovine trypsin; the arginine side-chain of the latter inserts into the specificity pocket in a "non-canonical" manner. The L-stereoisomer of 4-TAPAP, whose binding geometry towards trypsin was only poorly defined, is bound to the thrombin active-site in a compact conformation. In contrast to NAPAP, the distal p-amidino/guanidino groups of 4-TAPAP and MQPA do not interact with the carboxylate group of Asp189 in the thrombin specificity pocket in a "symmetrical" twin N-twin O manner, but through "lateral" single N-twin O contacts; in contrast to the p-amidino group of 4-TAPAP, however, the guanidyl group of MQPA packs favourably in the pocket due to an elaborate hydrogen bond network, which includes two entrapped water molecules. These thrombin structures confirm previous conclusions of the important role of the intermolecular hydrogen bonds formed with Gly216, and of the good sterical fit of the terminal bulky hydrophobic inhibitor groups with the hydrophobic aryl binding site and the S2-cavity, respectively, for tight thrombin active site binding of these non-peptidic inhibitors. These accurate crystal structures are presumed to be excellent starting points for the design and the elaboration of improved antithrombotics.


Three-dimensional structure in solution of acyl-coenzyme A binding protein from bovine liver. The three-dimensional structure of acyl-coenzyme A binding protein as encoded by the recombinant gene in Escherichia coli has been determined using nuclear magnetic resonance (n.m.r.) spectroscopy. The structure consists of four alpha-helices A1 (residues 3 to 15), A2 (residues 20 to 36), A3 (residues 51 to 60), and A4 (residues 65 to 85). A1 and A4, and A2 and A3, run in parallel pairs. A2 runs anti-parallel to A1 and A4. The three-dimensional structure of the protein is reminiscent of a shallow bowl with a rim. The "rim" is characterized by many polar and charged groups, whereas the inside and outside surface is predominantly hydrophobic with patches of uncharged polar hydroxyl groups of threonyl, serinyl and tyrosyl residues. The inside bottom contains through two epsilon-amino groups of lysine residues (Lys13 and Lys32) suggesting that the binding site for the nucleotide part of the acyl-coenzyme A part of the ligand molecule is at the inside surface of the bowl. The structure determination was done on the basis of measurements of the intensities of nuclear Overhauser effects (NOEs) and coupling constants that were translated into interatom distance restraints for 833 atom pairs, and 87 dihedral angle restraints, of which 23 were in chiral centers. In all, 42 hydrogen bonds were identified by n.m.r. and provided an additional 84 distance restraints. A total of 20 structures were calculated and the structures can be aligned to a root-mean-square deviation of 0.5 A for the backbone atoms of the residues in the four helices. A region of six residues could not be defined by the restraints obtained by n.m.r. The program Pronto was used for the spectrum analysis in general, and especially for the assignment of the individual NOEs, the integration of the cross peaks, and the measurements of the coupling constants. The programs DIANA and X-PLOR have been used in the structure calculations and evaluations.


Hydrogen bonding in globular proteins.

Hydrogen bonding in globular proteins. A global census of the hydrogen bonds in 42 X-ray-elucidated proteins was taken and the following demographic trends identified: (1) Most hydrogen bonds are local, i.e. between partners that are close in sequence, the primary exception being hydrogen-bonded ion pairs. (2) Most hydrogen bonds are between backbone atoms in the protein, an average of 68%. (3) All proteins studied have extensive hydrogen-bonded secondary structure, an average of 82%. (4) Almost all backbone hydrogen bonds are within single elements of secondary structure. An approximate rule of thirds applies: slightly more than one-third (37%) form i----i--3 hydrogen bonds, almost one-third (32%) form i----i--4 hydrogen bonds, and slightly less than one-third (26%) reside in paired strands of beta-sheet. The remaining 5% are not wholly within an individual helix, turn or sheet. (5) Side-chain to backbone hydrogen bonds are clustered at helix-capping positions. (6) An extensive network of hydrogen bonds is present in helices. (7) To a close approximation, the total number of hydrogen bonds is a simple function of a protein's helix and sheet content. (8) A unique quantity, termed the reduced number of hydrogen bonds, is defined as the maximum number of hydrogen bonds possible when every donor:acceptor pair is constrained to be 1:1. This quantity scales linearly with chain length, with 0.71 reduced hydrogen bond per residue. Implications of these results for pathways of protein folding are discussed.


Molecular structure of the B-DNA dodecamer d(CGCAAATTTGCG)2. An examination of propeller twist and minor-groove water structure at 2.2 A resolution. The crystal structure of the dodecanucleotide duplex d(CGCAAATTTGCG)2 has been solved to 2.2 A resolution and refined to an R-factor of 18.1% with the inclusion of 71 water molecules. The structure shows propeller twists of up to -20 degrees for the A.T base-pairs, although there is probably only one (weak) three-centre hydrogen bond in the six base-pair AT narrow minor-groove region. An extensive ribbon of hydration has been located in this groove that has features distinctive from the classic "spine of hydration". Solvation around phosphate groups is described, with several instances of water molecules bridging between phosphates.


Structure of RecA-DNA complexes studied by combination of linear dichroism and small-angle neutron scattering measurements on flow-oriented samples. By combining anisotropy of small-angle neutron scattering (SANS) and optical anisotropy (linear dichroism, l.d.) on flow-oriented RecA-DNA complexes, the average DNA-base orientation has been determined in RecA complexes with double-stranded (ds) as well as single-stranded (ss) DNA. From the anisotropy of the two-dimensional SANS intensity representation, the second moment orientation function S is obtained. Knowledge of S is crucial for the interpretation of l.d. spectra in terms of orientation of the DNA bases and the aromatic amino acid residues. The DNA-base planes are essentially perpendicular to the fibre axis of the complex between RecA and dsDNA in the presence of cofactor ATP gamma S. A somewhat tilted base geometry is found for the RecA-ATP gamma S complexes with single-stranded poly(dT) and poly(d epsilon A). This behaviour contrasts the RecA-ssDNA complex formed without cofactor which displays a poor orientation of the bases. Well-ordered bases in the ssDNA-RecA complex is possibly reflecting the role of RecA in preparing a nucleotide strand for base-pairing in the search-for-homology process. While the central SANS intensity is essentially independent of the pitch of the helical complex, a secondary intensity maximum, which becomes focused upon flow orientation, is found to be a sensitive measure of the pitch. The pitch values for the complexes compare well with cryo-electron microscopy results but are slightly larger than those seen for uranyl-stained samples.


Structure of DNA-RecA complexes studied by residue differential linear dichroism and fluorescence spectroscopy for a genetically engineered RecA protein. The structure of complexes of RecA with double-stranded and single-stranded DNA was studied by linear dichroism spectroscopy, fluorescence quenching and fluorescence anisotropy measurements. One of the two tryptophan residues (Trp291) of RecA was replaced by genetic engineering for an ultraviolet light-transparent threonine. This modified RecA protein shows, within experimental errors, the same DNA-binding kinetics and stoichiometry as the wild-type protein and no significant variation with respect to in vivo repair function was observed between cells with the two protein forms. By comparing the dichroic and fluorescence properties of the wild-type versus the modified protein, when bound to DNA, information about orientation and environment of the Trp291 chromophore in the complex could be obtained. The indole chromophore of Trp291Z was found to be oriented with its pseudo-long axis tilted 61 degrees and the aromatic plane is tilted 27 degrees relative to the fibre axis. Trp291 shows low mobility within the protein and therefore the deduced orientation may be used as a "handle" on the protein at the construction of three-dimensional models of RecA-DNA complexes. Comparison with the orientation for this residue in the crystal structure of the RecA homopolymer fibre indicates no measurable reorientation of the C-terminal subdomain of RecA upon DNA binding. Whereas the accuracy of the orientation determination of tryptophan, in absolute terms, is rather poor, changes of its orientation can be detected with high precision. Thus, similar Trp291 orientations are obtained in the complexes with single-stranded and double-stranded DNA, indicating similar structures of the protein fibres. The fluorescence quenching results indicate that the protein region of Trp291 is not involved in the binding of DNA.


Crystal structure of cleaved equine leucocyte elastase inhibitor determined at 1.95 A resolution. The crystal structure of active-site cleaved equine leucocyte elastase inhibitor, a member of the serpin superfamily, has been solved and refined to a crystallographic R-factor of 17.6% at 1.95 A resolution. Despite being an intracellular inhibitor with rather low sequence homology of 30% to human alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor, the three-dimensional structures are very similar, with deviations only at the sites of insertions and few mobile secondary structure elements. The better resolution in comparison with the structures of other cleaved serpins allows a more precise description of the so-called R-state of the serpins.


X-ray structure of the DNase I-d(GGTATACC)2 complex at 2.3 A resolution. The crystal structure of a complex between DNase I and the self-complementary octamer duplex d(GGTATACC)2 has been solved using the molecular replacement method and refined to a crystallographic R-factor of 18.8% for all data between 6.0 and 2.3 A resolution. In contrast to the structure of the DNase I-d(GCGATCGC)2 complex solved previously, the DNA remains uncleaved in the crystal. The general architecture of the two complexes is highly similar. DNase I binds in the minor groove of a right-handed DNA duplex, and to the phosphate backbones on either side over five base-pairs, resulting in a widening of the minor groove and a concurrent bend of the DNA away from the bound enzyme. There is very little change in the structure of the DNase I on binding the substrate. Many other features of the interaction are conserved in the two complexes, in particular the stacking of a deoxyribose group of the DNA onto the side-chain of a tyrosine residue (Y76), which affects the DNA conformation and the binding of an arginine side-chain in the minor groove. Although the structures of the DNA molecules appear at first sight rather similar, detailed analysis reveals some differences that may explain the relative resistance of the d(GGTATACC)2 duplex to cleavage by DNase I: whilst some backbone parameters are characteristic of a B-conformation, the spatial orientation of the base-pairs in the d(GGTATACC)2 duplex is close to that generally observed in A-DNA. These results further support the hypothesis that the minor-groove width and depth and the intrinsic flexibility of DNA are the most important parameters affecting the interaction. The disposition of residues around the scissile phosphate group suggests that two histidine residues, H134 and H252, are involved in catalysis.


Thermal motions of surface alpha-helices in the D-galactose chemosensory receptor. Detection by disulfide trapping. The D-galactose chemosensory receptor of Escherichia coli is a .32 kDa globular protein possessing two distinct structural domains, each organized in an alpha/beta folding motif. Helices I and X lie at adjacent approximately parallel positions on the surface of the N-terminal domain, near the hinge region. In order to analyze the relative thermal motions of these two helices, the present study utilizes a generalizable disulfide trapping approach: first, site-directed mutagenesis is used to place a pair of cysteine residues at locations of interest on the protein surface, then disulfide bond formation is used to trap intramolecular cysteine-cysteine collisions resulting from thermal motions. Specifically, four engineered di-cysteine receptors have been constructed, each possessing one cysteine at position 26 on helix I, and a second cysteine at varying positions on helix X. A fifth control receptor possesses one cysteine at position 26, and a second on the opposite surface of the molecule. These surface cysteine substitutions have little or no effect on the measurable receptor parameters as judged by ligand binding equilibria and kinetics, protein stability, and 19F nuclear magnetic resonance, indicating that the engineered receptors are useful probes of native backbone dynamics. Spatial and kinetic features of backbone motions have been investigated by measuring intramolecular disulfide formation rates for cysteine pairs in the fully liganded receptor. The resulting rates decrease monotonically with increasing distance between cysteines in the crystal structure, while no disulfide formation is observed for the control pair unless the molecule is unfolded. The minimum translational amplitudes of the observed backbone motions range from 4.5 to 15.2 A, and the minimum rotational amplitudes are as large as 35 degrees. For each motion the rate of intramolecular sulfhydryl-sulfhydryl collision has been estimated from the measured rate of disulfide formation: the 4.5 and 15.2 A translations yield approximately 10(4) and approximately 10 collisions s-1 molecule-1, respectively. These collision rates, which are faster than ligand dissociation, likely underestimate the actual motional frequencies since only an undetermined fraction of the total motions yield collisions. The simplest plausible trajectory capable of producing such collisions is a rate-limiting translation of one or both helices along their long axes, coupled with minor helix rotations. When sugar is removed from the receptor, a substantial increase in backbone dynamics is observed, indicating the presence of new long-range backbone trajectories. Overall, the results suggest that internal motions in proteins may have larger amplitudes than previously observed.


Contacts between Tet repressor and tet operator revealed by new recognition specificities of single amino acid replacement mutants. We have analyzed the DNA binding properties of Tet-repressor mutants with single amino acid residue replacements at eight positions within the alpha-helix-turn-alpha-helix DNA-binding motif. A saturation mutagenesis of Gln38, Pro39, Thr40, Tyr42, Trp43 and His44 in the second alpha-helix was performed; in addition, several substitutions of Thr27 and Arg28 in the first alpha-helix were constructed. The abilities of these mutant repressors to bind a set of 16 operator variants were determined and revealed 23 new binding specificities. All repressor mutants with DNA-binding activity were inducible by tetracycline, while mutants lacking binding activity were trans-dominant over the wild-type. All mutant proteins were present at the same intracellular steady-state concentrations as the wild-type. These results suggest the structural integrity of the mutant repressors. On the basis of the new recognition specificities, five contacts between a repressor monomer and each operator half-site and the chemical nature of these repressor-operator interactions are proposed. We suggest that Arg28 contacts guanine of the G.C base-pair at operator position 2 with two H-bonds, Gln38 binds adenine of the A.T base-pair at position 3 with two H-bonds, and the methyl group of Thr40 participates in a van der Waals' contact with cytosine of the G.C base-pair at position 6 of tet operator. A previously unrecognized type of interaction is proposed for Pro39, which inserts its side-chain between the methyl groups of the thymines of T.A and A.T base-pairs at positions 4 and 5. Computer modeling of these proposed contacts reveals that they are possible using the canonical structures of the helix-turn-helix motif and B-DNA. These contacts suggest an inverse orientation of the Tet repressor helix-turn-helix with respect to the operator center as compared with non-inducible repressor-operator complexes, and are supported by similar contacts of other repressor-operator complexes.


Crystallization and preliminary crystallographic characterization of GTP cyclohydrolase I from Escherichia coli. GTP cyclohydrolase I of Escherichia coli has been purified from a recombinant bacterial strain. The enzyme was crystallized from 0.6 M-sodium citrate and from 0.8 M-sodium/potassium phosphate, respectively. Crystals grown in citrate showed X-ray diffraction extending to a resolution better than 3 A. The space group was P2(1) with cell dimensions a = 204.8 A, b = 210.1 A, c = 72.2 A, alpha = gamma = 90 degrees and beta = 95.8 degrees.


Crystallization and preliminary crystallographic studies of glucosamine-6-phosphate deaminase from Escherichia coli K12. Hexameric glucosamine-6-phosphate deaminase from Escherichia coli has been crystallized isomorphously with both phosphate and ammonium sulphate as precipitants, over a wide pH range (6.0 to 9.0). The crystals belong to space group R32 and the cell parameters in the hexagonal setting are a = b = 125.9 A and c = 223.2 A. A complete native data set was collected to 2.1 A resolution. Self-rotation function studies suggest that the hexamers sit on the 3-fold axis and have point group symmetry 32, with a non-crystallographic dyad relating two monomers linked by an interchain disulfide bridge. A possible packing for the unit cell is proposed.


Purification, crystallization and preliminary crystallographic study of neuraminidase from Vibrio cholerae and Salmonella typhimurium LT2. The nanH genes of Vibrio cholerae and Salmonella typhimurium LT2 coding neuraminidase were cloned separately in Escherichia coli, and the expression products purified. Single crystals of the V. cholerae neuraminidase were obtained using the hanging drop vapour diffusion method with polyethylene glycol as precipitant at pH 7.2. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 71.9 A, b = 79.0 A, c = 165.7 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 2.5 A. Single crystals of the S. typhimurium neuraminidase were obtained by hanging drop with potassium phosphate as precipitant at pH 7.2. The crystals also belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 47.4 A, b = 82.8 A, c = 92.4 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 1.8 A.


Crystallization and preliminary X-ray diffraction studies of aspartic proteinase from Irpex lacteus. Crystals of ILAP (Irpex lacteus aspartic proteinase) have been obtained by the hanging drop method using ammonium sulfate as a precipitant. The crystals are monoclinic, space group P2(1) with cell dimensions a = 54.5 A, b = 79.6 A, c = 37.5 A, beta = 96.8 degrees. The crystals are quite stable to X-rays and diffract beyond 1.9 A resolution. There is one molecule in the asymmetric unit.


Primary structure of the second largest subunit of human RNA polymerase II (or B). The cDNA of the second largest subunit of RNA polymerase II (or B) from HeLa cells has been cloned and sequenced. A predicted amino acid sequence of 1174 residues (calculated molecular mass of 133,896 Da) was derived from the longest open reading frame and compared to the sequences of homologous subunits of polymerases of eukaryotic, archaeal and bacterial origin. After optimal alignment, about 16% of the residues were found to be conserved throughout evolution, from human to Escherichia coli. About 2/3 of the overall length of the conserved domains delineated by these residues are clustered within the C-terminal half of the human polypeptide, whereas the remaining is spread over its N-terminal half. The putative functional significance of these conserved domains is discussed.


Refined 2.5 A structure of murine adenosine deaminase at pH 6.0. The X-ray structure of murine adenosine deaminase complexed with the transition-state analogue 6-hydroxyl-1,6-dihydropurine ribonucleoside has been determined from a single crystal grown at pH 4.2 and transferred to mother liquor of increasing pH up to a final pH of 6.0 prior to data collection. The structure has been refined to 2.5 A to a final crystallographic R-factor of 20% using phases from the previously refined 2.4 A structure at pH 4.2. Kinetic measurements show that the enzyme is only 20% active at pH 4.2 whereas it is fully active between pH 6.0 and pH 8.5. The refined structures at either pH are essentially the same. Consideration of the pKa values of the key catalytic residues and the mechanism proposed on the basis of the structure suggests that the ionization state of these residues is largely responsible for the pH dependence on activity.


Protein-ligand energetics assessed using deoxy and fluorodeoxy sugars in equilibrium binding and high resolution crystallographic studies. Hydrogen bonding interactions are one of the most important single factors in protein-ligand interactions and molecular recognition. To probe the energetics of the interactions, we have analyzed the binding of 1-deoxy-, 2-deoxy- and 6-fluoro-6-deoxy- analogues of D-galactose (Gal) to a primary high-affinity periplasmic receptor for monosaccharide active transport. Kd values and atomic structures refined at 1.81 to 1.45 A resolution of the complexes have been determined and compared with those of Gal binding. With binding site residues and the bound modified sugars in nearly identical positions as found in the complex with Gal, the binding of 1-deoxy-Gal or 2-deoxy-Gal reflects the overall contribution of 1.8 kcal mol-1 per hydrogen bond (neutral-charge type) to the affinity of Gal. Neglected in these estimates is the contribution of van der Waals' forces that accompany the formation of hydrogen bonds with each sugar hydroxyl. Contrary to expectations, the 6-fluoro-6-deoxy analogue proved to be an inadequate probe of Gal OH6 as a hydrogen bond donor due to the binding of a new water molecule and structural changes arising from the electronegative fluoro group. This study sheds new light on the energetics of protein-ligand interactions and the use of engineered ligands in assessing these interactions.


Non-competitive inhibition of group I intron RNA self-splicing by aminoglycoside antibiotics. Aminoglycoside antibiotics inhibit self-splicing of group I intron RNA in vitro at concentrations as low as 10(-6) M. The sites of interaction and the mechanism of inhibition have yet to be determined. A comparative study of inhibition by different 2-deoxystreptamine analogues reveals structural features of the aminoglycoside antibiotics required for their interaction and effect on group I introns. Complete antibiotic inhibition of the two steps of splicing was not reversed at high concentrations of guanosine, indicating a non-competitive inhibition. A mutant group I intron in which the conserved guanosine nucleotide of the G-binding site had been replaced by an adenosine, was sensitive to the antibiotics providing direct evidence that the antibiotics do not interact with the G-binding site in the same way as the guanine base. In addition kinetic analyses of the self-splicing process in the presence of antibiotic inhibitors supported a non-competitive mechanism of the mixed type for inhibition of the ribozyme.


Cell-cycle control of a cloned chromosomal origin of replication from Caulobacter crescentus. Caulobacter crescentus cell division is asymmetric and yields distinct swarmer cell and stalked cell progeny. Only the stalked cell initiates chromosomal replication, and the swarmer cell must differentiate into a stalked cell before chromosomal DNA replication can occur. In an effort to understand this developmental control of replication, we employed pulsed-field gel electrophoresis to localize and to isolate the chromosomal origin of replication. The C. crescentus homologues of several Escherichia coli genes are adjacent to the origin in the physical order hemE, origin, dnaA and dnaK,J. Deletion analysis reveals that the minimal sequence requirement for autonomous replication is greater than 430 base-pairs, but less than 720 base-pairs. A plasmid, whose replication relies only on DNA from the C. crescentus origin of replication, has a distinct temporal pattern of DNA synthesis that resembles that of the bona fide C. crescentus chromosome. This implies that cis-acting replication control elements are closely linked to this origin of replication. This DNA contains sequence motifs that are common to other bacterial origins, such as five DnaA boxes, an E. coli-like 13-mer, and an exceptional A + T-rich region. Point mutations in one of the DnaA boxes abolish replication in C. crescentus. This origin also possesses three additional motifs that are unique to the C. crescentus origin of replication: seven 8-mer (GGCCTTCC) motifs, nine 8-mer (AAGCCCGG) motifs, and five 9-mer (GTTAA-n7-TTAA) motifs are present. The latter two motifs are implicated in essential C. crescentus replication functions, because they are contained within specific deletions that abolish replication.


Novel Escherichia coli mutant, dnaR, thermosensitive in initiation of chromosome replication. A newly isolated Escherichia coli mutant thermosensitive in DNA synthesis had an allele named dnaR130, which was located at 26.3 minutes on the genetic map. The mutant was defective in initiation of chromosome replication but not in propagation at a high temperature. This mutant was capable of growing in the absence of the rnh function at the high temperature by means of a dnaA-independent replication mechanism. In the mutant exposed to the high temperature, an oriC plasmid was able to replicate, although at a lower rate than at the low temperature. The plasmid replication at the high temperature depended on the dnaA function essential for the initiation of replication from oriC. The mutant lacking the rnh function persistently maintained the oriC plasmid at the high temperature in a dnaA-dependent manner. Thus, the dnaR function was required for initiation of replication of the bacterial chromosome from oriC but not the oriC plasmid. This result reveals that a dnaR-dependent initiation mechanism that is dispensable for oriC plasmid replication operates in the bacterial chromosome replication.


Video-enhanced microscopy of organelle movement in an intact epithelium. Digitally enhanced video microscopy has provided improved optical resolution in the study of intracellular organelle/particle movement, particularly in extruded axoplasm and certain thin single cell systems. We report here, for the first time, particle movement in an intact, isolated epithelium, the killifish proximal convoluted tubule. Cytoplasmic particles exhibited predominantly unidirectional linear movement approaching several microns in length, sometimes with multiple turns. The velocities of 34 particles measured in 11 cells averaged 0.29 microns/sec (range, 0.007-3.1 microns/sec). Microtubules--the well-established basis for organelle movement in cells--were present but were sparsely represented in electron micrographs of these cells. Video-enhanced microscopic techniques can now be applied to the study of organelle/particle movement in an intact epithelium.


Morphology of the antennae of two species of biting midge: Culicoides impunctatus (Goetghebuer) and Culicoides nubeculosus (Meigen) (Diptera, Ceratopogonidae). Scanning and transmission electron microscopy of the antennae of Culicoides impunctatus and Culicoides nubeculosus show that males and females share five sensillum types. Sensilla chaetica resemble mechanoreceptors, each innervated by a single neurone whose dendrite terminates distally in a tubular body: the arrangement of sensilla on male antennae suggests that females are located by sound. The antennae have both sharp- and blunt-tipped sensilla trichodea, sharp-tipped sensilla on only the distal third and blunt-tipped sensilla on all subsegments. These sensilla are typical of olfactory receptors, having multiporous walls and being innervated by a number of neurones with bifurcating dendrites ascending the hair shafts. Sensilla basiconica occur on the distal five subsegments of the female antenna and the distal three subsegments of the male antenna. Sensilla coeloconica always occur on subsegment one and sometimes on a number of other subsegments, depending on sex and species. Both basiconic and coeloconic sensilla have double walls and unbranched dendrites and may be either olfactory or thermo- and/or hygroreceptors. All antennae except those of male C. impunctatus antennae have sensilla ampullacea, apparently deep-seated olfactory or thermoreceptors. Small peg sensilla fitting the description of contact chemoreceptors occur only at the tip of the male antenna.


Intracytoplasmic ciliary elements in epidermal cells of Syndesmis echinorum and Paravortex cardii (Platyhelminthes, Dalyellioida). Epidermal cells of Syndesmis echinorum and Paravortex cardii contain many intracytoplasmic ciliary components: clusters of centrioles disorganized and incomplete short axonemes composed of loosely organized microtubules of irregular lengths, fully formed axonemes though some with fewer than nine doublets, and ciliary rootlets. Furthermore, conspicuous dense granules are found in solitary groups in the cytoplasm. Clusters of dense granules are also closely associated with Golgi complexes and developing axonemal microtubules. Since the dense granules decrease in number as the axonemes increase, it is likely that the granules are involved in the formation of axonemal microtubules. Ciliary elements are especially abundant in epidermal cells of Paravortex cardii embryos, some of them resembling those previously described by several authors in differentiating ciliated cells engaged in centriologenesis and ciliogenesis. Attention has been focused on the relative proportion and position of these elements, as well as the different morphology and several assembling states that they exhibit in epidermal cells of adult S. echinorum and adults and embryos of P. cardii. A functional interpretation of some of the findings is given, which allows us to suggest a sequence of ciliogenetic events that occur in epidermal cells of both species.


Retinoic acid enhances monoclonal antibody WE3 reactivity in the regenerate epithelium of the adult newt. Monoclonal antibody (mAb) WE3 recognizes an antigen that is developmentally expressed in the wound epithelium during adult newt limb regeneration. Experiments were designed to determine whether retinoic acid (RA), dissolved in dimethyl sulfoxide (DMSO) and administered by intraperitoneal injection, would enhance the temporal appearance of the WE3 antigen. RA given on days 1 or 4 after amputation, when the WE3 antigen is not yet detectable, resulted in moderate reactivity to mAb 2 days after injection and strong reactivity throughout the wound epithelium 4 days after injection. DMSO alone had no enhancing effect. RA also caused limb skin epidermis to exhibit reactivity to mAb WE3, initially near the amputation level, but then also more proximally. By 4 and 6 days after RA injection, epidermis of the flank, eye lid, and unamputated hind limbs also became strongly reactive to mAb WE3. Outer layers of skin epidermis were shed, resulting in an epidermis only one or two cells thick. Epidermis of newts given DMSO alone remained non-reactive to mAb WE3. When RA was given on days 7 and 10 after amputation, when a low level of mAb WE3 reactivity is already present in the wound epithelium, a considerable enhancement of mAb WE3 reactivity occurred through the next few days. No such enhancement was seen with DMSO alone. RA also greatly increased mAb WE3 reactivity in the wound epithelium of denervated limbs, in which case the wound epithelial reactivity to mAb WE3 is normally low. Retinol palmitate also increased mAb WE3 reactivity. The results raise the possibility that the WE3 antigen is a component of most if not all retinoid target tissues in newts.


Neuromuscular organization of feline anterior sartorius: II. Intramuscular length changes and complex length-tension relationships during stimulation of individual nerve branches. The feline anterior sartorius is a long strap-like muscle composed of short muscle fibers. Nerve branches that enter this muscle contain the axons of motor units whose constituent muscle fibers are distributed asymmetrically within the muscle. In the present study, twitch and tetanic isometric contractions were evoked by stimulating individual nerve branches while muscle force was recorded and intramuscular length changes were monitored optically by the movement of reflective markers on the muscle. Contractions elicited by stimulating the parent nerve produced little change in the positions of the surface markers. Contractions elicited by stimulating the proximally or distally directed nerve branches caused the muscle to shorten at the end closest to the nerve branch and lengthen at the opposite end. Some muscles were supplied by a centrally directed nerve branch whose stimulation produced variable effects: in some cases a portion of the muscle shortened whereas the rest lengthened, but in other cases, the positions of the surface markers showed little change. The intramuscular length changes produced by stimulating single nerve branches were greater during isometric contractions at short whole-muscle lengths than at long whole-muscle lengths. The twitch and tetanic length-tension relationships obtained by stimulating the individual nerve branches were not congruent with the length-tension relationship produced when the parent nerve was stimulated. At short whole-muscle lengths, stimulation of a single nerve branch generated only a small fraction of the force that could be generated by the muscle when the parent nerve was stimulated. As whole-muscle length increased, an increased fraction of total muscle force could be generated by stimulating a single nerve branch. The results suggest that a complex relationship between passive and active elements contributes to the total muscle force and depends on the distribution of active and passive muscle units throughout the muscle.


Programmed cell death and heterolysis of larval epithelial cells by macrophage-like cells in the anuran small intestine in vivo and in vitro. The degenerative processes in the larval small intestine of Xenopus laevis tadpoles during spontaneous metamorphosis and during thyroid hormone-induced metamorphosis in vitro were examined by electron microscopy. Around the beginning of spontaneous metamorphic climax (stages 59-61), both apoptotic bodies derived from larval epithelial cells and intraepithelial macrophage-like cells suddenly increase in number. The macrophage-like cells become rounded and enlarged because of numerous vacuoles containing the apoptotic bodies. Mitotic profiles of the macrophage-like cells, however, are localized in the connective tissue where different developmental stages of macrophage-like cells are present. After stage 62, the intraepithelial macrophage-like cells decrease in number, while large macrophage-like cells which include the apoptotic bodies and retain intact cell membranes and nuclei appear in the lumen. Degenerative changes similar to those during spontaneous metamorphosis described above could be reproduced in vitro. In tissue fragments isolated from the small intestine of stage 57 tadpoles and cultured in the presence of thyroid hormone, the number of intraepithelial macrophage-like cells reaches its maximum around the 3rd day of cultivation when the larval epithelial cells most rapidly decrease in number. These results suggest that the rapid degeneration of larval epithelial cells occurs not only because of apoptosis of the epithelial cells themselves but also from heterolysis by macrophages. The macrophages probably originate in the connective tissue, actively proliferate, migrate into the larval epithelium around the beginning of metamorphic climax, and are finally extruded into the lumen.


Ultrastructural and cytochemical aspects of oogenesis in Castrada viridis (Platyhelminthes, Rhabdocoela). The heterocellular female gonad of the typhloplanoid Castrada viridis consists of a single germarium and two rows of vitellaria. The germarium, composed of a germinative zone and a growth zone, is surrounded externally by a layer of accessory cells the function of which is hypothesized in this study. The main feature of oocyte differentiation is the synthesis of small electron-dense inclusions produced by the rough endoplasmic reticulum (R.E.R.) and Golgi complex. The electron-dense content of the egg inclusions reacts positively to the cytochemical test used to detect polyphenols and is only partially extracted following incubation in protease. The genesis, composition, and peripheral location of egg inclusions in mature oocytes suggest that they could represent residual eggshell granules. The presumed function of eggshell granules is discussed and their fine morphology is compared with that observed in other neoophoran Platyhelminthes.


Jaw-muscle activity in ferrets, Mustela putorius furo. Electromyographical (EMG) activity was recorded bilaterally from the masseter and temporalis muscles of alert ferrets (Mustela putorius furo) during mastication and crushing. Electromyographic activity was also recorded during biting while a bite-force transducer placed between the carnassial teeth registered forces ranging from 1.5 to 48.8 N. Linear regression analysis demonstrates that temporalis and masseter EMG activity are linearly related to bite force. Electromyographic activity from the balancing-side muscles is nearly equal to EMG activity of the working-side muscles during bone crushing with the carnassial teeth. It is hypothesized that a high percentage of balancing-side muscle activity in ferrets can be recruited during carnassial biting because the postglenoid process prevents ventral displacement of the working-side mandibular condyle.


Spatial and functional relationship between myocytes and fibroblasts in the rabbit sinoatrial node. In an attempt to understand better the directional differences in conduction velocity in the rabbit sinoatrial node, a possible conductive role of the abundant connective tissue surrounding the myocytes has been investigated. In particular, starting from the finding of communicating junctions between heart muscle cells and fibroblasts in tissue culture, heterologous gap junctions were searched for in thin sections of the rabbit sinoatrial node. Within and at the edge of nodal cell clusters, fibroblasts often show thin sheet-like extensions parallel to the surface of myocytes. In contrast to the intimately contacting myocytes, fibroblast extensions are kept separated from the myocytes by the basement membrane of the latter. Besides some rare undefined membrane appositions a single tiny gap junction-like structure was found between a fibroblast and a myocyte in a tissue area in which the calculated number of gap junctions between myocytes amounts from 1.10(4) to 3.10(4). Yet, fibroblasts are linked together regularly by small gap junctions containing a wider gap than the junctions between the myocytes (1.4 +/- 0.4 nm vs. 1.0 +/- 0.4 nm, resp., P less than 0.05). As an alternative to direct electrical coupling, the possibility of interaction between fibroblasts and nodal cells by capacitive coupling has been considered. Model calculations based on the reconstruction of some fibroblast extensions parallel to nodal cells show that the current which can be transmitted from discharging nodal cells to fibroblasts is negligible. It is concluded that fibroblasts do not participate in the impulse conduction within the sinoatrial node. The origin of the directional differences in conduction velocity in the sinoatrial node must be found in the spatial arrangement of the myocytes and the distribution of the gap junctions between these cells only.


Serum interleukin 6 levels become elevated in acute myocardial infarction. We have examined serum interleukin 6 (IL-6) levels in 12 patients with acute myocardial infarction (AMI). IL-6 levels became elevated in all patients, following the rise of serum creatine kinase (CK) activity. Peak IL-6 levels showed a good correlation with peak serum C-reactive protein (CRP) levels, while there was no direct relationship between peak IL-6 levels and peak CK activity. IL-6 mRNA was not detected in unstimulated "quiescent" rat cardiocytes cultured in serum-free medium, but its expression was induced by exposure of the cells to serum or ionomycin. These results show that IL-6 is synthesized in the myocardium and serum IL-6 levels become elevated in AMI, suggesting that IL-6 could affect the progression and/or healing processes of AMI.


Anti-ischaemic and endothelial protective actions of recombinant human osteogenic protein (hOP-1). Osteogenic protein one (hOP-1), a member of the transforming growth factor-beta (TGF-beta) supergenic family, was studied for its anti-ischaemic properties in rats subjected to myocardial ischaemia and reperfusion. Ten minutes after ligation (i.e., just prior to reperfusion) of the left coronary artery, 2 or 20 micrograms/rat recombinant human (hOP-1) or its vehicle, was given intravenously. hOP-1 at 20 micrograms significantly reduced reperfusion injury 24 h later compared to rats receiving only vehicle (i.e., 0.9% NaCl). hOP-1 was also found to preserve rat coronary endothelial function (i.e., release of endothelium-derived relaxing factor, EDRF) in perfused hearts following global ischaemia and reperfusion. Moreover, hOP-1 also significantly inhibited adherence of rat neutrophils to rat vascular endothelium in vitro. Thus, hOP-1 exerts significant anti-ischaemic effects. Some of this cardioprotection may be related to the ability of hOP-1 to preserve endothelial function and inhibit neutrophil adherence to the endothelium.


Cultured vascular endothelial cell susceptibility to extracellularly generated oxidant injury. To understand better the effect of oxidant injury on vascular endothelial cells, human saphenous vein endothelial cells were cultured at atmospheric (pO2 of 150 mmHg) or low (pO2 of 40 mmHg) oxygen tensions. The cellular rates of growth, antioxidant enzyme activities (superoxide dismutase, catalase, and glutathione peroxidase), phospholipid fatty acids and cellular susceptibility to extracellularly generated oxidants (hypoxanthine-xanthine oxidase) were measured. The antioxidant enzyme activities were regulated by oxygen tension and significantly differed by day 14. The cells cultured at the low oxygen tension had significantly (P less than 0.01) lower antioxidant activities than the cells cultured at the high oxygen tension. The cells cultured at an oxygen tension of 150 mmHg were more resistant to shrinkage and lipid peroxidation from the oxidants than the cells cultured at a pO2 of 40 mmHg by day 14. Since arterial and venous endothelial cells are perfused with blood at a pO2 of 100 and 40 mmHg, respectively, the postcapillary venous endothelial cells should have lower antioxidant enzyme activities than the precapillary arterial endothelial cells.


Calcium transients in isolated cardiac myocytes are altered by 1,1,1-trichloroethane. 1,1,1-Trichloroethane is a widely used solvent that is annually linked to several cases of sudden death following accidental exposure or abuse. Sudden death is believed to be due to ventricular fibrillation or myocardial depression. The purpose of this study was to investigate the mechanism of myocardial depression by assessing the influence of 1,1,1-trichloroethane on intracellular Ca transients in single neonatal rat ventricular myocytes using spectrofluorometric analysis of fura-2-Ca binding. Cells were exposed to 1,1,1-trichloroethane in Hanks' balanced salt solution aliquoted as a 0.2% DMSO solution by a single pass suffusion in an environmentally controlled chamber. 1,1,1-Trichloroethane (0.25 mM-8 mM) reduced the height of electrically (1 Hz, 60 V, 10 ms) induced Ca transients concentration dependently and reversibly to a maximum of about 50% with no effect on diastolic Ca concentration. Video motion analysis revealed an inhibition of contractility in the same concentration range. 1,1,1-Trichloroethane inhibited cytosolic Ca increase in response to KCl-induced (90 mM) depolarizations and further decreased the limited Ca transients in ryanodine (1 microM) pretreated myocytes. Increased external Ca (5 mM) antagonized the effect of 0.5 mM 1,1,1-trichloroethane on the Ca transients. 1,1,1-Trichloroethane reduced the caffeine (10 mM) releasable Ca pool in myocytes. These results show that 1,1,1-trichloroethane inhibits Ca mobilization during excitation-contraction coupling in ventricular myocytes. An inhibitory action on the influx of extracellular Ca as well as on sarcoplasmic reticulum Ca release and sequestration is likely to be responsible for this action.


Differential regulation of insulin-like growth factor I by growth hormone and thyroid hormone in the heart of juvenile hypophysectomized rats. Recent data suggest that the heart can act as both a source and target for the actions of polypeptide growth factors. Insulin-like growth factor I (IGF-I) is a polypeptide that has both mitogenic and differentiation properties that function at the autocrine/paracrine level, and has recently been demonstrated to be expressed in the heart. This knowledge, coupled with the observation that thyroid hormone (T3) promotes relative cardiac growth compared to the proportional increases in body and heart growth evoked by growth hormone (GH), lead us to speculate whether differential induction of cardiac IGF-I may account for the specialized trophic effects of T3 on the heart. Cardiac IGF-I gene expression was studied in an in vivo model in which cardiac growth in the hypophysectomized juvenile rat was stimulated with either GH, T3 or GH + T3. Two week infusions of T3 that resulted in cardiac growth, but no gain in body weight, resulted in a 4.6-fold increase in cardiac IGF-I mRNA levels compared to hypophysectomized controls. GH infusions that resulted in similar cardiac growth, but were accompanied by proportional body growth, had no effect on cardiac IGF-I mRNA levels. These data are the first to demonstrate stimulation of cardiac IGF-I mRNA levels by T3 and further support cardiac autocrine/paracrine actions for this polypeptide growth factor.


The effects of a PAF antagonist on ischemic myocardial damage and arrhythmia in the dog. Myocardial ischemia is associated with accumulation of lyso-phospholipids, including lyso-platelet activating factor, the degradation product and precursor of platelet activating factor. These compounds produce cellular and microvascular damage and, in the myocardium, depression of contractility and arrhythmia. The potent platelet activating factor antagonist, WEB 2086, or placebo, was infused (IV) 10 min before constriction of the proximal left anterior descending coronary artery in open-chest dogs. Two protocols were followed: the dose of WEB 2086 was 0.5 mg/kg in those subjected to 20 min ischemia with 10 min reperfusion (n = 40) and 5 mg/kg preceding 60 min ischemia alone (n = 24). There was no significant difference in the number of ventricular premature complexes between WEB 2086 and placebo treated dogs during either period of ischemia. On reperfusion in those surviving 20 min of ischemia, 5 of the 18 WEB 2086 and 9 of the 18 placebo treated dogs developed ventricular fibrillation (NS). After 60 min of myocardial ischemia, there was no statistical difference in histological changes (nuclear swelling, aggregation of chromatin, myofibrillar separation) between groups. Hence, no substantial effect of relatively large doses of WEB 2086 on ischemia-induced histological change or arrhythmia was found in this preparation.


Effects of oxygen free radicals on isolated cardiac myocytes from guinea-pig ventricle: electrophysiological studies. Free oxygen radicals are formed during early reperfusion and are thought to contribute to some types of reperfusion abnormalities, including arrhythmias and myocardial stunning. The purpose of this study was to investigate electrophysiological effects of oxygen free radicals using voltage clamped single ventricular myocytes from guinea-pig hearts. Oxygen free radicals were produced enzymatically by the direct addition of xanthine oxidase (XOD, 0.04 U/ml) in the experimental chamber to a solution containing hypoxanthine (0.96 mM). The generation of oxygen radicals was confirmed by the formation of adrenochrome from adrenaline. Oxygen radicals caused automaticity of isolated myocytes within 20-30 min, followed by later hypercontracture. The percentage of rod-shaped cells declined sigmoidally as a function of time, with a half maximal value at 40.9 +/- 1.6 min, and a Hill slope of -0.10 +/- 0.01 (n = 26). These effects were prevented by a combination of superoxide dismutase (10(5) U/L) plus catalase (10(6) U/L). The rate at which cells underwent morphological shape changes was unchanged by ryanodine (0.5 microM) which is thought to act on the sarcoplasmic reticulum or by the Ca2+ channel blockers nisoldipine (1 microM) or Cd2+ (30 microM). Cellular automaticity and hypercontracture were delayed by variable degrees, and sometimes completely prevented, by zero (1 mM EGTA) extracellular Ca2+, MnCl2 (2 mM) and LaCl3 (50 microM), and amiloride (1 mM). On the other hand, in the presence of a low extracellular Na+ (30 mM) or caffeine (10 mM), hypercontracture occurred at a faster time scale. Whole cell voltage clamping revealed a decrease of the inward rectifying K+ current (IK1), and a decrease of the peak of the L-type Ca2+ current (ICa,L). The total ICa,L during the clamp step was increased, mainly because of an increased time constant of inactivation (47.6 +/- 4.7 ms to 72.7 +/- 15.5 ms after 30 min, n = 4, P less than 0.05). We conclude that oxygen radicals cause automaticity and hypercontracture of isolated myocytes, that these effects may be due to an increased intracellular Ca2+ concentration ([Ca2+]i), and despite an increased ICa,L, that the enhanced Ca2+ influx may occur predominantly via the Na/Ca exchange.