Analysis of seminal plasma from roosters carrying the Sd (sperm degeneration) allele.

In previous research, subfertile roosters carrying the Sd (sperm degeneration) allele were characterized by malformed proximal efferent ducts. An abnormal biochemical milieu within the excurrent ducts of the testis was inferred. The objective of the present study was to compare seminal plasma composition between mutant (Sd) and normal (sd+) roosters. Phenotypic mutants were selected--on the basis of sperm viability--from a flock of Delaware roosters. After weekly ejaculations, sperm viability was < 60% for mutant roosters as compared to 100% for normal roosters. As reported previously, sperm viability in mutants increased to normal levels after frequent ejaculation. When comparisons were based on semen containing 100% viable spermatozoa, mutants were comparable to normal roosters with respect to sperm concentration and seminal plasma osmolality. Neither genotype was characterized by seminal plasma proteolytic activity at neutral pH. In contrast, seminal plasma from mutants was characterized by an imbalance of electrolytes, amino acids, and protein. Because the rooster lacks accessory sex glands, seminal plasma composition reflects excurrent duct function. Consequently, the abnormal seminal plasma composition of Sd roosters is attributed to excurrent duct dysfunction.


Thymosin alpha-1 enhances the fertilizing capacity of human sperm cell: implication in diagnosis and treatment of male infertility.

The effects of synthetic thymosin peptides (T alpha 1 and T beta 4) and their antibodies on the fertilizing capacity of human sperm cells were investigated. T alpha 1, but not the T beta 4, significantly (p < 0.001) increased the human sperm penetration rates in sperm penetration assay (SPA). Antibodies to both T alpha 1 and TB4, which predominantly bound to the acrosomal region of human sperm cell in the indirect immunofluorescence technique (IFT), also significantly (p < 0.001) increased (up to 4.7-fold) the human sperm penetration rates in SPA. The T alpha 1 and antibodies to both T alpha 1 and T beta 4 enhanced spontaneous as well as calcium ionophore-induced acrosome reaction and release of acrosin from the human sperm cells. There was no effect of T alpha 1 and antibodies to T alpha 1 and T beta 4 on percent sperm motility, although they significantly affected various motility characteristics such as velocity, amplitude of lateral head displacement (ALH), and beta frequency--the motility parameters involved in hyperactivation phenomenon of sperm cells. Both T alpha 1 and T beta 4 were detected in the seminal plasma of fertile men, and the levels of T alpha 1 were significantly (p = 0.002) lower in the seminal plasma of infertile men having defective sperm function. These results indicate that the thymosin molecules, especially T alpha 1, may have a role in human sperm capacitation leading to acrosome reaction. These findings also suggest that the T alpha 1 may find clinical applications in the specific diagnosis and treatment of male infertility in humans.


Immunocytochemical analysis of estrogen and progestin receptors in uteri of steroid-treated and pregnant cats.

The purpose of this study was to determine the distribution of estrogen receptors (ER) and progestin receptors (PR) in specific uterine cell populations during various steroid hormone treatment regimens, and to determine if ER and PR distribution in the uterus is altered during implantation and the establishment of pregnancy in the cat. The tissues were processed for indirect immunocytochemical localization of receptors using specific monoclonal antibodies against ER and PR. ER were present in the nuclei of all epithelial cells and stromal fibroblasts in endometrium obtained from ovariectomized animals, whereas PR were only detectable in the nuclei of stromal fibroblasts. There was an apparent increase in the staining intensity and number of nuclei that stained positively for both ER and PR in all cell populations after 14 days of estradiol treatment. The administration of progesterone for 14 and 21 days, in the presence or absence of continuous estradiol, reduced the apparent intensity of staining and the number of nuclei staining positively for both ER and PR. ER were undetectable in the luminal epithelium, but remained in the glandular epithelial cells and stromal fibroblasts, whereas PR were only detectable in stromal fibroblasts. ER and PR localization in the endometrium obtained from estrus animals was similar to that observed in the estradiol-treated animals. A general decrease in intensity of staining for both ER and PR was evident by Day 5 postcoitus in pregnant animals. This decrease in intensity of staining continued until Day 12 postcoitus, when the distributions of ER and PR were similar to those observed in the ovariectomized estradiol-primed, progesterone-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)


Melatonin decreases estrogen receptor expression in the medial preoptic area of inbred (LSH/SsLak) golden hamsters.

Daily late afternoon injections of melatonin (25 micrograms/day s.c.) were found to reduce the number of cells expressing estrogen receptor immunoreactivity in the medial preoptic area of ovariectomized inbred (LSH/SsLak) golden hamsters. Employing immunocytochemical analysis with the H222 monoclonal antibody to the human estrogen receptor, we examined the effects of melatonin on estrogen receptor expression in the hypothalamus, particularly the medial preoptic area, of ovariectomized virgin female hamsters. Analysis of the results showed that melatonin administration induced a 50-70% decrease in numbers of estrogen receptor-immunoreactive neurons in the medial preoptic area of ovariectomized female hamsters. Furthermore, an overall qualitative decrease in the intensity of estrogen receptor immunoreactivity was observed. In intact regularly cycling female hamsters used to monitor the efficacy of melatonin treatment, there were significant reductions in the serum levels of FSH, LH, and prolactin as measured by radioimmunoassay and in uterine and pituitary weights after 8 wk of melatonin treatment. These results suggest that melatonin may exert its anti-reproductive effects in hamsters by modulating estrogen receptor levels in medial preoptic area neurons, thus influencing steroid feedback mechanisms.


Missing generations of spermatocytes and spermatids in seminiferous epithelium contribute to low efficiency of spermatogenesis in humans.

Daily sperm production per gram parenchyma (DSP/g) in humans is only 25 or 35% of that for most species including rats and nonhuman primates. To explain the low efficiency of spermatogenesis in humans, the number of generations of germ cells (spermatocytes and spermatids) and the number of these germ cells in each generation were determined for each spermatogenic stage in men with varied efficiencies. Testes were obtained at autopsy, fixed by vascular perfusion with glutaraldehyde, further fixed in osmium, and embedded in Epon 812 before 0.5-micron sections were stained with toluidine blue. Tubular cross sections were photographed, and spermatogenic stages were determined by two observers. Testes were divided into three groups on the basis of DSP/g. The number of generations of spermatocytes and spermatids was greater (p < 0.05) in the high (2.01 +/- 0.05) and intermediate (1.77 +/- 0.04) than in the low (1.45 +/- 0.15) DSP/g group. All groups had a lower number of generations of spermatocytes and spermatids compared to the optimum value of three. The number of these generations per cross section was related (r = 0.85; p < 0.01) to DSP/g in these men. The number per cross section of spermatocytes, spermatids, and the combined number of germ cells was higher (p < 0.01) in the high than in the low DSP/g group. The combined number of germ cells per cross section was related (r = 0.85; p < 0.01) to DSP/g. The combined number of germ cells was higher in the high versus the low DSP/g group in stages I through V, but this difference was significant only in stages IV and V.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization and immunolocalization of bovine uterine retinol-binding protein.

Endometrial explants obtained from cows between Days 13 and 29 of pregnancy were cultured for 24 h in modified minimum essential medium in the presence of [35S]methionine or [3H]leucine. Proteins synthesized and released into medium were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Uterine luminal flushings were obtained from cyclic cows (Days 2-20 of estrous cycle) and early pregnant cows (Days 17-22). Endometrial tissues from cows on Days 17 and 29 of pregnancy were prepared for immunocytochemistry. A uterine secretory protein, which consisted of five isoelectric variants (pI 5.3-6.1) of identical molecular mass (23,000 Da), was shown to react immunologically with antiserum raised against bovine placental retinol-binding protein (bpRBP). Limited N-terminal sequence analysis of two major isoforms showed that the protein had nearly complete homology with bovine placental and plasma retinol-binding protein (RBP) over the first 25 amino acids. Through use of bpRBP antiserum, immunoreactive RBP was detected in uterine flushings collected from cows in the late luteal phase of the estrous cycle and early pregnancy by Western blotting, and in medium conditioned by uterine explants prepared at Days 13-29 of pregnancy by immunoprecipitation. Immunoreactive RBP was localized in endometrial surface and glandular epithelium on Days 17 and 29 of pregnancy by immunocytochemistry. These results demonstrate that RBP is a product of bovine uterine tissues. The uterine RBP may play an important role in vitamin A transport between maternal tissues and developing embryos.


Purine-induced block to mouse embryo cleavage is reversed by compounds that elevate cyclic adenosine monophosphate.

The second or third cleavage of mouse embryos from several strains of females is blocked in the presence of hypoxanthine. To begin to determine the mechanism of the block, we studied several aspects of cell metabolism in blocked embryos, including the stage of the cell cycle and the levels of transcription, translation, and protein phosphorylation. In addition, we attempted to reverse the block by transfer of cytoplasm, transfer of RNAs from dividing cells, and co-culture with compounds that elevate cAMP. Our results indicate the following: 1) that the embryos were blocked in interphase; 2) the expression of lac Z linked to SV40 promoter was depressed, but not blocked; 3) overall protein synthesis was depressed but the appearance of early embryonic proteins was not blocked; 4) overall phosphorylation of proteins was not affected; 5) microinjection of additional cytoplasm from non-blocking embryos did not reverse the block; and 6) compounds that elevate cAMP did reverse the block. Thus, the purines apparently do not prevent early embryonic gene expression or most phosphorylation events, but do inhibit a critical cell process occurring during interphase of the cell cycle that can be compensated by elevations in cAMP.


Preferential attachment of cock spermatozoa to the perivitelline layer directly over the germinal disc of the hen's ovum.

In vitro incubation of cock spermatozoa with perivitelline layer (PL) from recently ovulated ova of the hen resulted in binding of spermatozoa to the PL and activation of the acrosome reaction. A simple quantitative technique was developed for assessing these events. Following incubation of the PL (0.5 cm2 sections) with spermatozoa, the PL section was rinsed and stained with Schiff's reagent. Microscopic examination revealed holes in the PL that were assumed to be sites of spermatozoa penetration. Utilizing this technique, a correlation was demonstrated between sperm concentration and the number of spermatozoa attaching to the PL and undergoing an acrosome reaction. Pre-treatment of spermatozoa with solubilized PL inhibited spermatozoa binding to pieces of intact PL. The PL overlying the germinal disc and a similarly sized section of PL from another area of the ovum were removed and incubated separately with spermatozoa (1 x 10(5) sperm/100 microliters). Spermatozoa showed preferential attachment and digestion of the PL from the germinal disc area (809 sperm/mm2) as compared to PL from other areas of the ovum (608 sperm/mm2). Spermatozoa attached to the PL in a circular, doughnut-shaped fashion in the area directly over the germinal disc.


Characterization of deoxyribonucleic acid synthesis and the transition from maternal to embryonic control in the 4-cell porcine embryo.

These studies were conducted to identify the point during the 4-cell stage at which the porcine embryo begins to control development. Reproductive tracts of gilts were flushed 48 h after the onset of estrus to obtain 1- and 2-cell embryos. To determine the duration of the 4-cell stage in vitro, development of 29 embryos was timed from cleavage to the 4-cell stage and from cleavage to the 8-cell stage. The average duration of the 4-cell stage was 50.5 h. The duration of the 4-cell stage was positively correlated (p < 0.01) with culture time in vitro before cleavage to the 4-cell stage. DNA content was determined by using the Feulgen's reaction and quantified with micro-densitometry. Staining units (SU; density x area) were calculated at 0, 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, and 36 h post-cleavage to the 4-cell stage (P4C). Results revealed a possible G1 phase (< 2 h) with DNA synthesis starting within 2 h P4C. DNA synthesis was completed by 16 h P4C, and was followed by an extended G2 phase. Embryos were evaluated for uptake and incorporation of [35S]methionine and for qualitative changes in protein profiles specific to time points during the 4-cell stage (2, 10, 14, 16, 18, 24, 30, and 40 h P4C). Methionine uptake and incorporation into protein followed similar patterns, both decreasing until 16-18 h P4C, followed by a steady increase through the 4-cell stage. Protein profiles revealed qualitative changes beginning at 14 and 16 h P4C.(ABSTRACT TRUNCATED AT 250 WORDS)


Purification and characterization of a human follicular fluid lipid transfer protein that stimulates human sperm capacitation.

Identification of the mechanisms responsible for sperm capacitation has been an active area of research for nearly four decades. Changes in the lipid composition of the sperm membrane is one of the biochemical events that occurs during sperm capacitation. We have been studying physiological effectors of some of these changes and have identified lipid transfer activity in fractions of human follicular fluid that stimulates sperm penetration of zona-free hamster oocytes. We report here the purification of a lipid transfer protein by sequential chromatography from human follicular fluid. This protein was purified greater than 20,000-fold for lipid transfer activity and greater than 28,000-fold for sperm penetration-inducing activity. This 64,000 molecular weight protein has a pI of approximately 5.0 and shares physicochemical characteristics with the plasma lipid transfer protein, LTP-I. Antibodies to LTP-I also recognize this protein and depletion of LTP-I from human follicular fluid by immunoaffinity chromatography renders the follicular fluid incapable of stimulating sperm penetration. We conclude that purified LTP-I is able to stimulate human sperm capacitation and that LTP-I is a molecule responsible for this stimulation in follicular fluid.


Survival of mouse morulae vitrified in an ethylene glycol-based solution after exposure to the solution at various temperatures.

Mouse morulae were exposed in one step to a vitrification solution (EFS, a modified PBS containing 40% ethylene glycol, 18% Ficoll, and 0.3-M sucrose) at various temperatures, then cooled rapidly in liquid nitrogen, and then warmed rapidly. All of the embryos exposed to the EFS solution for 0.5 min at 25 degrees C before vitrification developed in culture. However, survival rates were lower if the duration of exposure was prolonged to 2, 5, or 10 min. At lower ambient temperatures (20, 10, and 5 degrees C), high survival rates were associated with longer exposure to the EFS solution. The toxicity of the EFS solution was also lower at lower temperatures. The toxic injury of morulae was manifested as decompaction of the blastomeres. Among the three additives in the EFS solution, ethylene glycol, which can cross cell membranes, was responsible for the toxicity. The results show that the optimum time for exposure of the embryos to the EFS solution before rapid cooling varies with the ambient temperature, i.e., 0.5 min at 25 degrees C, 0.5-5 min at 20 degrees C, 2-5 min at 10 degrees C, and 2-10 min at 5 degrees C. If they are exposed for an optimum period, almost all mouse morulae can survive vitrification (94-100%).


Stress reduces the quality of gametes produced by rainbow trout.

In this study we have used the rainbow trout as a model animal to study the biological consequences of stress in terms of gamete quality and quantity. Groups of 30 mature male and female rainbow trout were subjected to repeated acute stress during the 9 mo prior to spawning. Time of ovulation, fecundity, and egg size were recorded in mature females, and sperm counts were carried out on the milt from the male fish, from both the stressed and control groups. Eggs from ovulated females were fertilized with milt from males subjected to the same treatment regime. Approximately 300 eggs from each female were fertilized with a sperm dilution of 10(-3) in diluent. Subsequent development of the fertilized eggs was then monitored. There were no differences in somatic weight or length between the two groups at the end of the experiment, but exposure of rainbow trout to repeated acute stress during reproductive development resulted in a significant delay in ovulation and reduced egg size in females, significantly lower sperm counts in males, and, perhaps most importantly, significantly lower survival rates for progeny from stressed fish compared to progeny from unstressed control fish. Hence, stress reduces the quality of gametes produced by rainbow trout.


Role of prostaglandins in the testosterone-dependent wolffian duct differentiation of the fetal mouse.

Recent observations from this laboratory indicated a role of prostaglandin E2 (PGE2) in masculine differentiation of the external genitalia of the fetal mouse, induced by fetal testosterone. In this communication, we further investigated the role played by PGE2 in the testosterone-induced differentiation of the internal genital tract (Wolffian duct) of the fetal mouse. Using in vitro organ culture bioassay of Wolffian duct differentiation, we determined the effect of a PG-depleting agent, namely, anti-PGE2 antibody, and of inhibitors of PG synthesis for their ability to prevent Wolffian duct differentiation in the presence of testosterone. We demonstrated that anti-PGE2 antibody inhibited Wolffian duct differentiation in a dose-dependent manner in embryonic male explants containing fetal testes. At 1:10 dilution, the antibody inhibited the appearance of the entire Wolffian duct as well as growth of the specimen. At 1:100 dilution, however, only development of the Wolffian duct was prevented, as indicated by the absence of regions of the Wolffian duct or by the presence of epithelial disintegration throughout the ductal lumen. The antibody at 1:1000 dilution produced no significant effect on the appearance of the Wolffian duct. PGE2 (10 micrograms/ml) replacement in the medium prevented Wolffian duct disintegration induced by anti-PGE2. We next determined whether the testosterone-dependent Wolffian duct differentiation requires ongoing PG synthesis within the reproductive tract and analyzed the effects of the compounds inhibiting PG synthesis at the level of phospholipase A2 (PLA2) namely, cortisone and dexamethasone-and of those inhibiting at the level of cyclooxygenase-namely, aspirin and indomethacin. We have demonstrated that both PLA2 and cyclooxygenase inhibitors inhibited Wolffian duct differentiation in the male explant, i.e., in the presence of testis. These compounds also prevented the appearance of the Wolffian duct in female explants induced by exogenous testosterone. PGE2 added in the medium blocked the anti-masculinizing effects of PG synthesis inhibitors both in the male and female specimens. Thus, it appears that PG synthesis plays a role in the testosterone-induced masculine differentiation of the Wolffian duct.


Estrogen- and progesterone-dependent secretory changes in the uterus of the sheep.

This study was undertaken to determine the effects of 17 beta-estradiol (E) and progesterone (P) on polypeptide synthesis and release from the uterus of the sheep. Uterine flushings (UF) and endometrium were obtained from ovariectomized untreated animals, ovariectomized animals treated with E (approximately 5-10 pg/ml) for 6 days (6E) and ovariectomized animals primed with E for 6 days then treated with P (approximately 1.5-3 ng/ml), in the continued presence of E, for an additional 6 days (6EP). Endometrium was cultured (24 h) in the presence of 3H-leucine (3H-leu) or 3H-glucosamine (3H-glcN), and newly synthesized and released proteins were detected in culture media by fluorography of 10% SDS gels. The quantity of proteins in UF and radiolabeled proteins in explant culture media did not change between treatment groups (p < 0.05). Qualitative changes in the synthesis and release of proteins were observed depending on the steroid treatment. An M(r) 57,000 protein was present in UF and 3H-leu-labeled culture media obtained from animals treated only with E and an M(r) > 200,000 was present in 3H-leu-labeled culture media of endometrium obtained from 6E and 6EP animals. An M(r) 44,000 protein was present only in UF from 6EP animals but could not be detected in endometrial culture media from animals undergoing this steroid treatment. These data show that the endometrium of the ovariectomized sheep undergoes alterations in secretory protein patterns which depend on the presence of E and P.


Characterization of the kinin system in the ovary during ovulation in the rat.

Ovulation has been noted for some time to bear a remarkable similarity to an inflammatory response. One of the principal components that is activated and helps mediate the events during an inflammatory response is the kinin system. Therefore, the purpose of the present study was to examine whether this system could be similarly activated and involved in the cascade of events that leads to ovulation. To answer this question, immature 23-day-old female rats were primed with eCG (10 IU) and ovulation was induced by administration of hCG (10 IU) 48 h later. Groups of rats were killed at 0 h, 10 h, 20 h, and 30 h after hCG for determination of ovulation, ovarian steroid levels, and changes in the levels of kinin system components. Plasma total kininogen levels did not change during the entire period studied. In contrast, ovarian total kininogen levels rose from 0 h to reach a peak at 10 h--a time immediately preceding the beginning of ovulation--after which the levels fell at 20 h, only to rise again at 30 h. Three species of kininogens, high molecular weight (HMW), low molecular weight (LMW), and T-kininogen, were shown to be present in the ovary. T-kininogen was the major kininogen present in the ovary, accounting for 60-92% of the total kininogen at any given time point during the ovulatory process. HMW kininogen levels accounted for only 1.2% of the total ovarian kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)


Patterns of intracellular Ca2+ concentrations in fertilized bovine eggs.

Sperm-induced calcium (Ca2+) changes were examined in zona pellucida-intact, mature bovine eggs injected with the fluorescent Ca2+ indicator fura-2 dextran (fura-2 D). Fifty four percent (37/68) of the dye-injected, inseminated bovine eggs were fertilized and 43% (16/37) of the fertilized eggs exhibited Ca2+ elevations during the time of measurement. All (16/16) of the eggs with Ca2+ elevations were fertilized but none of the unfertilized eggs (0/31) showed intracellular Ca2+ elevations. Six of 13 eggs that were later examined and found to be fertilized at the time of the Ca2+ recordings did not show sperm-induced Ca2+ elevations. Fifty percent (8/16) of the eggs with Ca2+ elevations exhibited a single Ca2+ rise as a response to sperm penetration during the 60-min period in which these eggs were monitored. Twelve percent (2/16) of the eggs responded with two Ca2+ elevations spaced by 50- and 51-min intervals and 38% (6/16) of the eggs exhibited multiple elevations with intervals of 15-29 min. In the latter group, one egg was polyspermic. The mean amplitude of the sperm-induced Ca2+ elevations was 564 +/- 58 nM. Eggs with single elevations reached higher peak concentrations than eggs with multiple elevations (p < 0.05). The mean duration of the Ca2+ elevations was 166 +/- 13 sec and was similar among eggs with different Ca2+ patterns. The first elevations detected occurred at a mean of 6.6 +/- 0.5 h after insemination. Fertilization in this study was confirmed by looking at pronuclear formation 16 h post-insemination or by DNA staining immediately after the fluorescence readings. Eggs exhibiting Ca2+ elevations ranged in stage of fertilization from just penetrated to pronuclear. Injection of inositol 1,4,5 trisphosphate (5 microM in the injection pipette) into 6 bovine eggs induced an immediate Ca2+ elevation with a mean peak Ca2+ value of 700 +/- 60 nM and a mean duration of 103 +/- 21 sec. Incubation of bovine eggs with 200 microM thimerosal induced periodic Ca2+ rises. The mean number of Ca2+ elevations observed in 35 min of recordings was 3.0 +/- 0.5 (n = 9, range 1-5). The mean peak Ca2+ value of the first thimerosal-induced Ca2+ elevation was 990 +/- 210 nM. The results of this study indicate that fertilization can evoke intracellular Ca2+ elevations in bovine eggs and that the periodicity of these Ca2+ elevations is different among eggs. Furthermore, both inositol 1,4,5-trisphosphate and thimerosal were able to induce intracellular Ca2+ release in bovine eggs.


Tissue uptake of human sex hormone-binding globulin and its influence on ligand kinetics in the adult female rat.

The distribution of human sex hormone-binding globulin (hSHBG) and its influence upon the kinetics of its ligands were assessed in the adult female rat, which lacks a comparable protein in serum. Purified hSHBG was administered i.v. to adult female rats as a single bolus. The plasma disappearance rate of immunoreactive hSHBG had one component with a half-life of 15 h. The estradiol (E2) binding activity of serum attributable to hSHBG was elevated 2-fold; during a continuous infusion of E2, hSHBG increased E2 serum levels above those of control infused animals. Treatment with hSHBG did not modify the plasma clearance of endogenous E2, but accelerated the disappearance rate of 5 alpha-dihydrotestosterone (DHT). In animals injected with a tracer dose of radioactive steroids, pretreatment with hSHBG increased uterine and oviductal accumulation of E2- but not DHT-associated radioactivity. This effect was specific to some E2 target tissues since hSHBG did not alter the concentration of E2- or DHT-associated radioactivity in the hypophysis, liver, diaphragm, or brain. Treatment with anti-E2 antibodies, which elevated E2 binding activity in serum, decreased the accumulation of E2-associated radioactivity in uterus and oviduct. Immunofluorescent localization of hSHBG revealed intense labeling of the uterine and oviductal epithelium. We conclude that this foreign hormone-binding globulin introduced in serum at concentrations that have minimal circulating reservoir effect on E2 can reach intracellular domains and affect the concentration of this ligand in target tissues.


Source of immunoreactive inhibin in the chicken ovary.

High concentrations of immunoreactive inhibin were detected in the plasma of the laying domestic hen using a heterologous RIA validated for use in the chicken. Cessation of egg production induced by restricting the intake of nutrients decreased circulating inhibin to approximately 20% of its original concentration within 8 days, indicating that the ovary is the major source of the measured material. Dissection of ovarian follicles revealed that inhibin is nearly exclusively produced in the granulosa cell layer. When expressed per milligram cell protein the concentration of inhibin decreased significantly in granulosa layers of follicles of succeeding order in the hierarchy (F4 to F1). The concentration of progesterone increased in the granulosa layers of the same follicles whereas oestradiol in the surrounding theca layers decreased. In vitro culture of granulosa cells derived from follicles at different stages of development confirmed the decrease in inhibin secretion as a function of follicular growth observed in vivo. The granulosa cell inhibin secretion is stimulated by LH as well as by FSH, the former being the most effective one. The physiological significance of these changes in inhibin concentration during follicular maturation requires further investigation. It may be concluded, however, that the chicken presents a useful model for the study of the endocrine as well as the paracrine function of ovarian inhibin.


Dietary fish oil delays puberty in female rats.

Marine oils contain eicosapentaenoic acid, a fatty acid that competes for cyclooxygenase and reduces the synthesis of dienoic prostanoids including prostaglandin E2 (PGE2). Since PGE2 plays an important role in the estrogen-stimulated release of hypothalamic GnRH on proestrus, it was postulated that a diet containing fish oil would delay first ovulation through inhibitory effects on GnRH release. Thirty, 22-day-old female Sprague-Dawley rats were fed a diet containing fish oil ad libitum. Controls were pair-fed an identical diet with the substitution of safflower oil as the dietary fat. All rats were killed on the morning of first metestrus after vaginal opening and the display of an estrous smear(s). Fish oil feeding did not affect growth as indicated by the lack of an observed effect on body weights or femur lengths. On the other hand, pituitary, ovarian, and uterine weights were significantly lower in the rats fed fish oil (p < 0.001). The age at first estrus of the rats fed fish oil was significantly increased compared with the controls (42.9 +/- 1.0 vs. 36.1 +/- 0.3 days; p < 0.001), whereas the number of rats with corpora lutea (CL), as well as the number of CL per ovary (2.3 +/- 0.4 vs 4.8 +/- 0.6 for controls; p < 0.001) was significantly reduced by fish oil feeding. GnRH concentration in the preoptic area/hypothalamus was significantly increased in the fish oil-fed rats (21.4 +/- 4.0 pg/mg vs. 7.6 +/- 2.2 pg/mg for controls; p < 0.01); radioimmunoassable hypothalamic PGE2 was concomitantly reduced (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


Study of blood compatible polymers. II. poly(N,N-disubstituted) acrylamides.

Poly(N,N-disubstituted) acrylamides with both hydrophilic and hydrophobic groups as substituents were synthesized. Different degrees of hydrophilicity were achieved by varying the bulk of the hydrophobic substituent. N-alkyl, N-(2-hydroxyethyl) acrylamides with alkyl substituents propyl (PROPAAm), octyl (OCTAAm) and benzyl (BENAAm) were synthesized. The swelling capacity of the polymers decreased with increase in bulk of the hydrophobic substituent. In vitro studies showed that the surfaces of these polymers did not induce platelet aggregation. Cell compatibility of these polymers was assessed by following the growth of human umbilical cord fibroblast cells. Pronounced cell growth and spreading was observed on the surfaces of polyOCTAAm and polyBENAAm. The relatively low cell growth on polyPROPAAm was ascribed to its high water content.


Macrophage response to microtextured silicone.

Seven different silicone surface textures were tested for effect on macrophage spreading and metabolic activity in vitro. Variables of the textured arrays that could modify spreading were determined to be the size, spacing between, depth, density, and orientation of the individual surface events and the roughness of the surfaces. Cells were influenced by the size of the events and the roughness of the surfaces more than any other variables. Cell morphology data, surface area and perimeter, could be divided into discrete regions that correlated well with the size of the events. Cell dimensions on 5 microns textures were smallest while those on smooth silicone and glass surfaces were the largest. Surface texture events may be modifying contact guidance of the cells or interacting with specific transmembrane proteins to alter cell shape and function. The mitochondrial activity of cells attached to the textured silicones was determined by measuring the amount of reduced MTT directly through live cells. Cells on polystyrene (PS), 5VP and 8VP textures were metabolically more active than cells on the other textures. PMA was used to stimulate cells on the various textures. PMA-stimulated cells, on the smaller textures, 2VP, 5VP and 5CP, were less active than test cells that were not stimulated. The inability of PMA to stimulate these cells may be due to a structural alteration of protein kinase C. An hypothesis is introduced that includes a possible mechanism of how a micrometre-sized surface texture could modify cell function.


Woven carbon surface replacement in the knee: independent clinical review.

An independent assessment was performed of the results of woven carbon implants of mesh or rods into articular cartilage defects performed by two surgeons in the knees of 96 patients. Eight had symptomatic osteochondritis, 31 had chondromalacia patellae and 57 had early osteoarthritis. They were reviewed up to 5 yr after operation. Seventy-six patients (79%) experienced an improvement in analogue pain score and similar improvements occurred in a range of functional activities (P = 0.002). No evidence of inflammatory change or deterioration in joint damage was found. It is concluded that this relatively minor procedure is valuable in younger patients with troublesome symptoms due to early articular cartilage damage and does not prejudice further operative treatment.


3-Methoxybutylcyanoacrylate: evaluation of biocompatibility and bioresorption.

The biocompatibility and bioresorption of 3-methoxybutylcyanoacrylate (MBCA) was evaluated in vivo using female Wistar albino rats. MBCA was found to elicit slight to moderate tissue reaction similar to isobutylcyanoacrylate (iBCA) which has been sold commercially as a surgical adhesive (Bucrylate, Ethicon). MBCA was judged less reactive to tissue than ethylcyanoacrylate (ECA). The MBCA implants in rat gluteal muscles also resorbed within approx. 16 wk while iBCA implants remained essentially unchanged at 36 wk in vivo. In vitro resorption in phosphate buffer (pH 7.2) at 37 degrees C showed the same trend. The MBCA performed similarly to iBCA as a haemostat on excised rat livers and as an adhesive on rat skin incisions and had comparable adhesive bond strength.


Biocompatibility of lipid microcylinders: effect on cell growth and antigen presentation in culture.

The authors are developing a lipid-based microcylinder for the controlled release of biological response modifiers and as templates for cellular migration and differentiation. These structures are comprised of a photopolymerizable phosphatidylcholine (1,2-ditricosa-10,12-diynoyl-sn-glycero-3-phosphocholine) and form spontaneously as a result of a thermotropic phase transition in aqueous solution or in a cosolvent solution of 70:30 ethanol:water. The hollow cylinders are helically wrapped lipid bilayers, variable in length (50-250 microns, depending on conditions of formation) and are 0.5-1.0 microns in diameter. The interaction has been examined of three types of lipid microcylinders: (1) monomeric, (2) photopolymerized by exposure to 254 nm light, and (3) surface-modified by incorporation of 6 mol% gangliosides, with different human cell lines and peripheral blood leucocytes to evaluate the biocompatibility of these structures. The proliferative status of U937 (a histiocytic monocyte), K562 (an erythroleukaemic cell), and Jurkat's derivative (a T-lymphoblast) as measured by pulsed tritiated thymidine was unaffected by the presence of up to 100 micrograms/ml of lipid microcylinders after 3 d in culture. Adherent human peripheral blood monocytes were shown to form adhesive contacts with the lipid microcylinders. An 'association' index from this interaction shows that after 3 d in culture, the association was much lower for those microcylinders that had incorporated ganglioside compared with monomeric or polymerized structures. The lipid microcylinders do not activate T-cells isolated from human peripheral blood, nor do they inhibit the activation of T-cells by phorbol esters or other mitogens.(ABSTRACT TRUNCATED AT 250 WORDS)


Body distribution of fully biodegradable [14C]-poly(lactic acid) nanoparticles coated with albumin after parenteral administration to rats.

Fully biodegradable polylactic acid (PLA) nanoparticles (90-250 nm) coated with human serum albumin (HSA) were prepared by high-pressure emulsification and solvent evaporation, using the protein as surfactant. A new analytical tool was developed, based on Mie's law and size exclusion chromatography, to establish that, after evaporation of the solvent, the protein saturates the surface of the nanoparticles, masking the PLA core. According to this technique, no HSA is encapsulated in the polymer matrix. A radiolabelled [14C]-PLA50 was synthesized to follow the fate of this new drug carrier after i.v. administration to rats. The time necessary to clear the albumin-coated nanoparticles from the plasma was significantly longer than for the uncoated ones but not extended enough to target cells other than mononuclear phagocytes. As deduced from whole-body autoradiography and quantitative distribution experiments, the 14C-labelled polymer is rapidly captured by liver, bone marrow, lymph nodes, spleen and peritoneal macrophages. Nanoparticle degradation was addressed following 14C excretion. The elimination of the 14C was quick on the first day (30% of the administered dose) but then slowed down. In fact, if the metabolism of the PLA proceeds to lactic acid which is rapidly converted into CO2 via the Krebs cycle (80% of the total excretion was fulfilled by the lungs), anabolism from the lactic acid may also have taken place leading to long-lasting radioactive remnants, by incorporation of 14C into endogenous compounds.


Adsorption of plasminogen from plasma to lysine-derivatized polyurethane surfaces.

The adsorption of plasminogen, the principal protein of the fibrinolytic pathway in blood, to a number of solid surfaces from plasma was investigated. This study forms part of a larger project to develop a fibrinolytic surface for blood-contacting applications. Polyurethanes incorporating lysine residues were developed in an attempt to promote selective adsorption of plasminogen from plasma through lysine-binding sites in the plasminogen molecule. The adsorption of plasminogen to these surfaces as well as to glass, 'conventional' polyurethanes and precursor sulphonated polyurethanes was investigated. Adsorption from citrated human plasma diluted with isotonic Tris buffer (pH 7.4) was measured under static conditions at room temperature using radioiodinated plasminogen. The following trends were observed. (1) Adsorption increases monotonically with increasing plasma concentration and there is no suggestion of transient adsorption (Vroman effect) on any of the surfaces studied. (2) Sulphonate groups appear to have a strong effect on plasminogen adsorption as was found previously for adsorption from buffer. (3) The lysine-derivatized material having the highest lysine content may show a slight increase in plasminogen binding affinity compared to its sulphonated precursor.


FT-IR measurement of emulsified cationic DMAEMA-MMA adsorption to calcium alginate.

The rate of adsorption of aqueous emulsions of DMAEMA-MMA copolymers to calcium alginate substrates was examined using ATR FT-IR spectroscopy. This work was undertaken as part of the development of a hybrid artificial pancreas. Aqueous DMAEMA-MMA copolymer emulsions containing < 25 mol% DMAEMA were slowly adsorbed to calcium alginate substrates; emulsions of 50 mol% DMAEMA-MMA copolymer were adsorbed rapidly to calcium alginate gels. The adsorption of 50 mol% DMAEMA-MMA copolymer emulsions to calcium alginate gels was attributed to electrostatic rather than simple van der Waals' attractive forces.


Multiple births: trends and patterns in Canada, 1974-1990.

This paper examines Canadian trends and patterns in multiple births in relation to total confinements, singleton births, maternal age, parity, gestational age and birth weight using vital statistics from 1974 to 1990. Multiple-birth rates in Canada increased from 912.8 to 1,058.9 per 100,000 confinements between 1974 and 1990. The increase is especially noticeable for women over 30. The rate of triplet and higher-order births increased from 8.3 to 21.7 per 100,000 confinements between 1974 and 1990. The proportion of multiple-birth babies that were pre-term (< 37 weeks gestation period) increased from 32.8% in 1974 to 45.8% in 1990. Factors associated with the increase in multiple births may include the use of assisted pregnancy techniques, and the fact that women aged 30 and older, who are at higher risk of a multiple birth, and who postponed their child bearing, have increased their fertility. The sharp increase in multiple-birth rates has implications for maternal and child health and health care costs.


Level of care for deinstitutionalized psychiatric patients.

The New York state Level-of-Care Survey (LOCS) was used in 1987-88 to assess the community care and social support needs of 936 mentally disabled community residents in Halifax, Nova Scotia. The cases were assigned an appropriate level-of-care by the computerized algorithm based on an 101-item questionnaire covering all aspects of the cases' physical and mental condition, needs and activities. Compared with their current setting, the algorithm assigned 60% of cases to the same level-of-care, 17% to a lower level, and 23% to a higher level-of-care. The net effect on social service costs of the reassignments would be a 7% increase. The increased costs for some cases ($1.5 million) would be partly offset by lowered costs for others ($900,000). The gradual shifting of 14 cases from the relatively expensive rehabilitation unit to more independent living at lower levels of care would compensate for most of the increases.


A statistical chronicle of tuberculosis in Canada: Part II. Risk today and control.

This report marks over 50 years of publication by Statistics Canada of annual reports on Tuberculosis Statistics. These years have witnessed what has been described as a conquest of tuberculosis in Canada. To quote George Jasper Wherrett in the Miracle of the Empty Beds: One hundred years ago the word consumption (as tuberculosis was then called) struck horror in human hearts. Today, in the western world, it barely evokes any emotion save a too easy surprise that it still exists. This statistical chronicle of tuberculosis in Canada is divided into two parts. Part I: From the Era of Sanatorium Treatment to the Present pulls together data from yellowed-with-age reports on tuberculosis and vital statistics, historical accounts, and modern computer files, to document the changes in tuberculosis incidence and mortality over past decades to the present. Part II: Risk Today and Control takes a closer look at those most vulnerable to contracting tuberculosis. It also looks at the future, the need for maintaining and, indeed, strengthening vigilance, and the work yet to be done to eradicate tuberculosis in Canada. The fight against tuberculosis is far from over.


The consistency of various high blood pressure indicators based on questionnaire and physical measures data from the Canada Health Survey.

Several indicators of the presence of high blood pressure, taken from different parts of Canada Health Survey data, are compared at the individual respondent level to determine their consistency and their usefulness, alone or in combination, in measuring the prevalence of hypertension. The results demonstrate some systematic and substantial discrepancies among various high blood pressure indicators derived from questionnaire data, and between questionnaire data and physically measured blood pressure status.


The National Population Health Survey: highlights of initial developments.

A National Population Health Survey is being developed for implementation across Canada in 1994. A description is given of the objectives, plans and current status of the survey. The current assumptions concerning the survey methodology are also outlined along with an early indication of the content.


Molecular computing for edge-enhanced laser imaging.

In order to illustrate the self-assembly capability, we consider a laser imaging experiment on a wet film that is made of bacteriorhodopsin (BR) molecules suspended in a diffusion-limited viscous medium. BR wet film is similar to a wet photograph film but having a finer resolution and adaptive pixel locations due to laser-induced thermal diffusion. The synergism between thermal diffusion of BR molecules (induced externally by a write-laser) and molecular photochromism (generated internally by a read-laser) is exploited naturally for edge-enhanced image applications.


Do biomolecules process information differently than synthetic organic molecules?

This paper compares information/signal processing in synthetic and biological molecules. The role of conformation-based (shape-based) mechanisms and electrostatic interactions in molecular recognition is discussed. In biological electron transfer, the 'electron shuttle'-mediated mechanism is contrasted with the mechanism based on pre-formed 'electron wires'. While biological information processing is thought to be more distributed (less discrete), an example of molecular switch is presented: visual transduction. We further speculate that visual transduction may be implemented in the form of a switch based on electrostatic interactions. The concept of intelligent materials is discussed with the well-known Bohr effect of hemoglobin oxygenation. Based on these examples, we argue that there are no fundamental differences between synthetic and biological molecules in their mode of information processing. In the pursuit of novel paradigms of molecular information processing, we also perceive no conflicts in developing molecular devices that emulate the switching function of conventional microelectronic devices.


Purple membrane multilayers: detailed structure and photoelectric characteristics of bacteriorhodopsin.

The structure of the Langmuir purple membrane (PM) monolayer was studied by scanning tunneling microscopy (STM). The topography of both the external and cytoplasmic sides of the monolayer was investigated. The thickness of the PM monolayer was determined to be about 4 nm. PM films include specific oval 'crater'-like structures of 50-60 nm in diameter.


Bacteriorhodopsin: a picosecond optoelectric signal transducer.

This paper summarizes the results found in our laboratory investigating the ultrafast light-induced charge separation in bacteriorhodopsin. A special technique was elaborated for dried oriented samples of long term stability. An upper limit of 21 ps was found by a direct electric method for the early charge separation processes. A permanent electric field on the surface of illuminated samples was demonstrated. The potential application of such samples as ultrafast optoelectric signal transducers is discussed.


Biosensors and enzyme immobilized electrodes.

A biosensor is a device which consists of a biological sensing element connected to a transducer. The transducer can be electronic, optical, electrical, etc. This emerging technology offers us a powerful tool which is radically altering our approach to analytical methods. It was realised that enzymes are natural sensors on account of their highly selective nature. Much of the impetus to the work has come from medical requirements. Instant analysis of clinical samples has an obvious appeal to physicians and patients alike. Of particular interest is the possibility of continuous 'in-vivo' monitoring of metabolites, drugs and proteins using miniature, portable systems. In recent years, there has been a growing demand for biosensors in the fields of veterinary science, animal husbandry, the food industry and environmental monitoring. However, the possibility of successful application rests upon future developments. Increasing attention will have to be paid to the engineering of both the basic components and the device on the whole. New biochemical reactions will either have to be discovered or engineered through genetic manipulation or chemical techniques. Optimization of response time, selectivity, stability and low costs should receive priority considerations.


Molecules as electronic components?

Aspects of electronics at the molecular level are reviewed. Molecules can store information in their different states. To transmit information, they need to interact with other molecules, but this affects the states. Excitation transfer is also more complex than sometimes realised. Information processing is best treated in terms of the whole system of interacting molecules, so that molecules cannot be treated simply as small conventional electronic components.


Molecular optical memories and switches based on photochromic dihydropyridines.

Photochromic behavior of dihydropyridines and the mechanism of the photochromism based on phenyl transfer is described. Formation of polar coloured species, changes in optical absorption and in the tau electron character of the molecular skeleton allow for the fabrication of molecular optical memories and switches.


Towards a biomolecular computer.

A new version of computing and information processing devices may result from major principles of information processing at molecular level. Non-discrete biomolecular computers based on these principles seems to be capable of solving problems of high computational complexity. One of the possible ways to implement these devices is based on biochemical non-linear dynamical systems. Means and ways to materialize biomolecular computers are discussed.


The seed germination model of enzyme catalysis.

The activity of enzymes and other biological macromolecules is often sensitively dependent on physiochemical context. Seed germination provides an analogy that helps to elicit the control and information processing capabilities of enzymatic networks. Like a seed, the enzyme takes a particular action (complexes with a specific substrate and catalyzes a specific reaction) when a specific set of milieu influences is satisfied. The context sensitivity, specificity and speed are enormously enhanced by the parallelism inherent in the electronic wave function (i.e. by the superposition of electronic states). This parallelism is converted to speedup through electronic-conformational interactions. The quantum speedup effect allows biological 'switches' to have qualitatively greater pattern recognition capabilities than electronic switches. Consequently the information processing and control capabilities of biomolecular systems exceed the capabilities obtainable from classical models and exceed the intuitive expectations that have developed through the study of such models.


Semantic commitments as a mode of non-programmable computation in the brain.

The natural language processor in the brain can cope with non-programmable computation. The average number of different lexical meanings per word serves as a quantitative figure in terms of which the extent of being non-programmable can be evaluated. The possible maximum average number of different lexical meanings per word that the brain of the subject reading the text can cope with while comprehending the context is found to be 3.3 with its standard deviation 0.15, beyond which the brain can no more succeed in comprehending the context. In contrast, the maximum average number of different lexical meanings per word that would make lexical disambiguation programmable is e = 2.718. Natural language processing in the brain is non-programmable in the sense that the manageable average number of different meanings per word is greater than e, but does not exceed roughly 3.3.


Magnetic fields stimulate peripheral nerve regeneration in hypophysectiomized rats.

The effect of a sinusoidal 50 Hz magnetic field, 0.4 mT on regeneration of the rat sciatic nerve was studied. The sciatic nerve of intact and hypophysectomized rats was crushed and regeneration was evaluated by the pinch test and by immunocytochemical staining for neurofilaments. The animals were exposed to the magnetic field for 3-6 days, between a pair of Helmholtz coils. Hypophysectomy of the rats resulted in an impaired regeneration. However, the magnetic field exposure stimulated regeneration in both the intact and hypophysectomized animals. The results imply that the pituitary gland is not involved in magnetic field induced stimulation of nerve regeneration.


Testosterone fails to rescue motoneurons from axotomy-induced death in young rats.

This study was undertaken to determine whether testosterone acting on muscle alone could enhance motoneuron survival after axotomy. Facial nerves were transected unilaterally in 10-day old rats in which androgen receptors were already present in skeletal muscle but not yet developed in motoneurons. Testosterone treatment lessened chromatolysis but failed to reduce neuronal loss which reached more than 50% 2 weeks postaxotomy. The inefficacy of testosterone was attributed to the death of motoneurons before they could re-establish synaptic contact with targets, thereby rendering target-derived trophic substances stimulated by testosterone unable to rescue motoneurons in a timely manner. The present finding is consistent with our hypothesis that functional neuromuscular connectivity is a prerequisite for testosterone to promote neuronal survival.


Axon outgrowth from grafts of human embryonic spinal cord in the lesioned adult rat spinal cord.

Adult rats with acute partial lesions of their upper thoracic spinal cords were implanted bilaterally with cell suspensions of 6-7 week-old embryonic human spinal cord tissue one segment above or below the lesions. After 14-19 weeks, the animals were perfusion-fixed and the tissue analysed with a light microscope after immunocytochemical labelling with an antiserum recognizing human, but not rat, intermediary neurofilaments. Using this method, extensive efferent projections were demonstrated extending longitudinally from the grafts into the host spinal cord, both in the caudal and rostral directions. Within the white matter tracts, dense bundles of fibres extended for about 3-4 mm, and single fibres were identified up to 10 mm away from the implants. Axonal growth of this length within host white matter has not previously been observed from intraspinal grafts of rat CNS tissue.


Expression of odorant receptors in spatially restricted subsets of chemosensory neurones.

From a rat olfactory library a cDNA clone (OR37) which is supposed to encode an odorant receptor protein has been isolated and characterized. Specific antisense RNA and in situ hybridization techniques have been employed to monitor the olfactory epithelium for the distribution of olfactory neurones expressing the OR37-gene. The OR37-transcripts were detected only in a subset of receptor cells segregated in two restricted areas of the olfactory epithelium. The clusters of reactive cells appear symmetrically in both nasal cavities. Within a reactive region only a subset of the cells expressed the receptor. The segregation of neurones expressing a distinct receptor supports the notion that a spatial component may be involved in coding odour quality.


Simultaneous EEG 10 Hz desynchronization and 40 Hz synchronization during finger movements.

Nineteen-channel EEG was recorded with closely spaced electrodes overlaying the left sensorimotor cortex during self-paced, voluntary right finger movements. Three right-handed people served as subjects. The EEG was analysed in the 10 Hz band (10-12 Hz) and in four 40 Hz bands (34-36, 36-38, 38-40, 40-42) by calculation of ERD time courses and ERD maps, whereby a ERD is characterized by a movement-related band power decrease. In all three subjects a close to C3 localized 10 Hz ERD was found, starting about 2 s prior to movement onset and continuing during movement. Along with this 10 Hz ERD a localized and short-lasting (about 0.5 s) burst of 40 Hz oscillations was embedded around movement onset. This can be interpreted as indicating that planning of movement is accompanied by a desynchronization of central mu rhythm and a generation of 40 Hz oscillations.


Synaptic origin of rhythmic visually evoked activity in kitten area 17 neurones.

Rhythmic patterns in neuronal activity in response to moving stimuli were observed in 28% of cells recorded extracellularly or intracellularly in area 17 of 4-16 week old anaesthetized and paralysed kittens. In both recording modes, oscillation frequencies ranged between 7 and 71 Hz, and were confined for 88% of cells in the 7-20 Hz band of the spectrum. A comparative study of firing autocorrelograms) and subthreshold activity (autocorrelation functions) indicates that the regularity of discharge stemmed from visually evoked oscillations of membrane potential at the same frequency. These oscillations are shown to result from extrinsic excitatory activity, since their amplitude, but not their frequency, depends on the resting membrane potential. The dependency on stimulus configuration supports the hypothesis that oscillations in neuronal output are dictated by periodic activity in afferent circuits selectively recruited by different attributes of the visual input which are not exclusively processed at the cortical level.


Development of subpopulations of GABAergic neurons in cat visual cortical areas.

The development of GABAergic interneurons in feline striate area and extrastriate areas was tracked by single and double labeling immunohistochemistry using antibodies to GABA and to molecular markers which identify subpopulations of GABAergic neurons in adult mammalian neocortex; i.e., neuropeptide Y, somatostatin, and the calcium-binding proteins parvalbumin and calbindin. The density of GABA-ir neurons was relatively constant during development and among visual areas. By contrast, most of the GABA-subpopulations increased in the cortex of visual areas during postnatal development, and thus the proportion of GABA-ir neurons which also expressed another molecular marker increased during development. By the end of the first postnatal month, the neurotransmitter phenotypes of the neocortical GABAergic neurons are mature.


bFGF induces its own gene expression in astrocytic and hippocampal cell cultures.

Basic FGF mRNA induction by bFGF was investigated in cell cultures from rat brain, i.e. postnatal day 2 cortex and embryonic day 18 hippocampus. In situ hybridization shows that after bFGF treatment (10(-10) M) for 14 h neurones and glial cells show a remarkable increase in bFGF mRNA production. Incubation of astrocytes with antisense bFGF phosphorothioate oligodeoxynucleotides (bFGF-PTOs) resulted in an inhibition of both bFGF induced and serum induced proliferation. The results indicate that bFGF is capable of inducing its own mRNA production. This induction, i.e. new synthesis of bFGF mRNA, seems to be essential for the mitogenic effect of both bFGF and serum components.


Quantitative age-related changes on nuclear invaginations of neocerebellar Purkinje cells.

The aim of this study was to provide stereological data for seeking whether Purkinje neurones increase their nuclear envelope invaginations with age. The nuclear parameters considered were: ratio of absolute surface area to volume, surface density and absolute surface area. The first two parameters did not significantly change, but the absolute surface area exhibited marked variations. Regression studies showed no significant linear correlations between any of the parameters and age; so, the increase in number and/or in depth of the invaginations was not suggested. The association of invaginations with a nuclear cap of Nissl substance is a constant feature, stressing that the physiological importance of the complex is maintained throughout the ageing process.


Familial influence on plaque formation in the beagle brain.

Aged canines exhibit central neuropathological changes strikingly similar to those seen in patients with Alzheimer's disease. In this study, brain tissue from pure bred beagles raised in a controlled environment were examined for Alzheimer-like pathology. The mean age of the animals was 15.6 years. The incidence of plaques among these 29 dogs was 65.5%. Of the 19 samples that demonstrated Alzheimer-like pathology, 18 were characterized as diffuse and one as neuritic. Plaque density was found to be independent of age. Plaque numbers were highest in the perirhinal cortex and the adjacent temporal cortex. Familial influence on plaque development is supported by congruence within 15 of the 16 litters examined (p < 0.001). In this environmentally controlled group the diffuse plaques were rarely converted to the dense neuritic plaques found in Alzheimer's disease.


Regional cerebral blood flow and P3 variables in patients with multiple lacunar infarcts.

We measured regional cerebral blood flow (rCBF) and event-related potentials (ERPs) in 36 patients with multiple lacunar infarcts and 14 age-matched normal subjects. rCBF was measured by the 133Xe inhalation method. ERPs were recorded during visual discrimination tasks using three kinds of stimuli. The patients showed lower mean cortical blood flow than normal subjects especially for the frontal cortex. Nontarget P3 latency in patients was longer than in normal subjects, while no significant differences could be found in target P3 latency between the two groups. Nontarget P3 latency correlated with frontal CBF. These results show that frontal lobe dysfunction may be particularly marked with multiple lacunar infarcts and suggest that reduction of frontal CBF is related to the impairment of the automatic processing associated with the nontarget P3 component.


Additivity of nicotinic and muscarinic cholinergic stimulation of substantia nigra.

Microinjection of cholinergic agonists into the anterior substantia nigra increases behaviours for which there is a pre-existing tendency and a low current rate. Rats received microinjections of carbachol, nicotine or a combination of both into the anterior substantia nigra. Carbachol and nicotine both stimulated consumption of palatable food in a dose-dependent manner (F(3,24) = 3.99, p < 0.02 and F(3,33) = 7.07, p < 0.01 respectively). Addition of carbachol to each dose of nicotine caused a significant increase in feeding compared with nicotine alone (F(1,23) = 7.01, p < 0.015). This suggests muscarinic or nicotinic cholinergic stimulation of the anterior substantia nigra can have behaviourally similar effects and, at the doses used here, the effects of nicotinic and muscarinic stimulation are additive.


Adenosine-induced hyperpolarization is depressed by glibenclamide in rat CA1 neurones.

The effect of the adenosine triphosphate (ATP)-sensitive potassium (KATP) channel blocker, glibenclamide, on adenosine-induced postsynaptic hyperpolarization was studied by means of intracellular recording techniques in TTX-treated CA1 neurones in the rat hippocampal slice. Glibenclamide applied in the CSF perfusion fluid at 30 microM reversibly depressed the 2-chloroadenosine-induced hyperpolarization and the increase in the membrane conductance. It is suggested that adenosine induces the opening of potassium channels in the postsynaptic membrane of CA1 neurones, including KATP channels in the mammalian central nervous system (CNS).


Astrocyte maturation and susceptibility to ischaemia or substrate deprivation.

We demonstrate that immature astrocytes die much earlier than mature astrocytes when substrate is deprived in the presence of oxygen. In contrast, mature astrocytes die much earlier than immature astrocytes when substrate is deprived in the absence of oxygen. These observations correlate well with the recent findings that the newborn rodent brain is more susceptible to damage from lack of substrate than from lack of oxygen. The basis for this susceptibility of immature astrocytes to substrate deprivation in the presence of oxygen is not clear, but perhaps is related to the ability of the cell to scavenge free radicals.


Developmental changes in distribution of NMDA receptor channel subunit mRNAs.

In situ hybridization analyses have revealed drastic changes in expression and distribution of five subunit mRNAs of the mouse NMDA receptor channel during brain development. The epsilon 1 subunit mRNA is expressed postnatally and widely in the brain. On the other hand, the epsilon 2 subunit mRNA is found throughout the entire embryonic brain, but its expression becomes restricted to the forebrain at postnatal stages. The epsilon 3 subunit mRNA appears postnatally and predominantly in the cerebellum, whereas the epsilon 4 subunit mRNA is abundantly expressed in the diencephalon and the brainstem at embryonic and neonatal stages. In contrast, the zeta 1 subunit mRNA distributes ubiquitously in the brain throughout development. These findings suggest that changes in the subunit composition of the NMDA receptor channel take place during brain development.


Auditory sensitivity in rats exposed to toluene and/or acetyl salicylic acid.

The present study investigates whether exposure to acetyl salicylic acid (ASA) affects the permanent auditory loss, caused by simultaneous exposure to toluene. Rats were exposed to toluene by inhalation (1,000 ppm), or to ASA by gavage (100 mg kg-1), or to both toluene and ASA, during 10 days. Toluene exposure caused a loss of auditory sensitivity recorded as auditory brainstem response, and no loss was found after exposure to ASA alone. The group exposed to both ASA and toluene had a more severe sensitivity loss (p < 0.05) at all frequencies compared with all other groups immediately after (2-5 days) exposure and also 4 months later. The results indicate that ASA permanently potentiates toluene induced loss of auditory sensitivity.


Representation of abstract attributes of auditory stimuli in the human brain.

Representations of abstract attributes of auditory stimuli in the human brain were demonstrated using the mismatch negativity (MMN), an event-related potential component elicited by a change in a repetitive sound. Stimuli were pairs of sinusoidal tones. There were two types of tone pairs in each block, standard (p = 85%) and deviant pairs (p = 15%), delivered in a random order. Standard and deviant tone pairs differed only in the direction of within-pair frequency change. In addition, the frequency levels of both the standard and deviant pairs varied randomly within a wide range in a block; thus the standard pairs shared the direction of the within-pair frequency change but not the absolute frequency level. Correspondingly, the deviant pairs only shared the opposite direction of the within-pair change. Nevertheless, the deviant tone pairs elicited MMN, implying that even the direction of the within-pair frequency change of the standard stimuli, and not just their absolute frequencies, developed a neural representation.


Novel anticonvulsant action of chronic melatonin in gerbils.

Melatonin, a hormone from the pineal gland, was tested for its anticonvulsant effects in male gerbils. Daily administration of melatonin (25 micrograms injection-1, s.c.) for ten weeks reduced the number and severity of seizures (total convulsion score: 7.67 +/- 1.83 in controls vs 2.47 +/- 0.90 in melatonin-injected animals, p < 0.05) associated with the injection of the convulsant, pentylenetetrazol (PTZ, 60 mg kg-1, s.c.). However, neither 12 weeks of short photoperiod exposure (LD 10:14) nor biweekly administration of melatonin pellets altered PTZ-induced convulsions. Overall, melatonin-injected gerbils were better able to survive and respond to seizures than control animals. No melatonin-injected gerbils died during seizure induction (0/31) while 5 out of 33 control gerbils died after PTZ injection. The mechanism for melatonin's anticonvulsant effects could be due to a direct inhibitory action on neural activity or a conversion of melatonin to an anticonvulsant compound that resembles the kynurenines.


Theoretical and methodological aspects of BCG vaccine from the discovery of Calmette and Guérin to molecular biology. A review.

The BCG vaccine has been used to prevent tuberculosis since 1921 and applied for immunostimulation in neoplasia since the 1960s. Both the preventive and immunostimulation effects have been evaluated and communicated with contradictory, positive and negative conclusions. For an objective evaluation and interpretation of the protective efficacy, effectiveness and efficiency of the BCG vaccination it must be considered that: (1) several BCG substrains have been developed in manufacturing laboratories that differ in the residual virulence which determines immunogenicity and reactogenicity; (2) various liquid and freeze-dried BCG vaccine production methods are used, resulting in different BCG viable units per dose; (3) quantitative bioassay methods are not yet being used for statistical quality control of the vaccine; (4) BCG products are applied in various demographical, epidemiological and socioeconomic conditions with different vaccination policies; (5) inadequate biostatistical models are often used to analyse efficacy, effectiveness and adverse reactions. The same conditions influence the precise evaluation of BCG immunostimulation in neoplasia. Recombinant DNA technology will modify production methods, and explain at the molecular level the mechanism of the protective effects BCG confers in tuberculosis and immunostimulation in neoplasia. High level laboratory techniques and biostatistical methods, based on probability logic and inductive inference, ensure appropriate experimental designs and the exact analysis of laboratory data and the results of vaccination policies. They will lead to the evaluation of the protective effect of BCG in order to reduce the BCG contradictions.


A comparative study of the polymerase chain reaction and conventional procedures for the diagnosis of tuberculous pleural effusion.

Preliminary reports by ourselves and others suggest that amplification of mycobacterial DNA by the polymerase chain reaction (PCR) is a sensitive and rapid diagnostic test for tuberculosis. We recently described a PCR assay with a 336 bp repetitive sequence specific for Mycobacterium tuberculosis as the DNA target, which gave encouraging results in culture-positive smear-negative clinical specimens. In the present prospective study of patients with pleural effusions we compared PCR of the pleural fluid with conventional procedures. 84 adult patients with pleural effusions were divided into 4 groups. In group A (44 patients), M. tuberculosis was detected by culture of pleural fluid, pleural biopsy or extrapleural source. In group B (6 patients), tuberculous infection was confirmed by histology (group A excluded). Group C (3 patients) had clinical evidence of tuberculosis. Group D (31 patients) had no evidence of active M. tuberculosis infection. Analysis of the pleural fluid confirmed a sensitivity for PCR of 81%. The sensitivity of pleural fluid culture, culture of pleural biopsy, and histology of biopsy was 52.8%, 69.8% and 77.3% respectively. There were however 7 PCR positive results within group D; 6 of these were in patients with malignant effusions. We conclude that for the diagnosis of M. tuberculosis PCR is more sensitive than laboratory culture as determined by the analysis of pleural fluids. Positive PCR results among patients with malignant effusions may be false-positives or the result of latent tuberculous infections. PCR should remain an investigational procedure until prospective studies in high and low prevalence areas have critically evaluated the specificity of the assay.


Virulence of Mycobacterium tuberculosis for guinea pigs: a quantitative modification of the assay developed by Mitchison.

One of the more accepted methods of assay of virulence of tubercle bacilli is one developed by Mitchison in which guinea pigs were infected by the intramuscular route with 1.0 mg of tubercle bacilli freshly harvested from Lowenstein Jensen medium and in which virulence was based on a subjective score of the extent of gross disease in the animal 6 weeks after infection. Due to the practical difficulties involved in such an assay when routinely performed, the following modifications were made: frozen stocks of the culture were prepared prior to the date of infection, the inoculum was quantitated by means of colony forming units, and virulence was based on the number of tubercle bacilli recovered from the spleen of infected animals 6 weeks after infection. A significant correlation was obtained between the findings from the Mitchison assay of virulence and the modification. The latter assay is recommended for its quantitative and objective features.


Studies on cell-wall deficient non-acid fast variants of Mycobacterium tuberculosis.

While the host-parasite relationship in tuberculosis still remains incompletely understood, there has been recent renewed interest in indications that tubercle bacilli are converted into metabolically inactive, non-acid fast (NAF) granular forms in the presence of host defence mechanisms and antituberculosis drugs. The present study investigates the mechanism of induction of these NAF variants in vitro and in vivo, and their ultimate pathogenicity. Evidence is provided that appears to clearly indicate that acid-fast mycobacteria are converted into NAF, cell wall deficient variants which remain dormant, only to revert to the parent, acid-fast bacilli in immune-compromised hosts, thence ultimately producing disease. It is then suggested that this may be one of the causes of the observed persistence of the bacilli in hosts in spite of chemotherapy. In a typical study in experimental animals in the present investigation, NAF variants were separated from lung lavage by differential centrifuging. When these were then injected into animals made immune-deficient with Freund's adjuvant or cyclophosphamide, they reverted to parent acid-fast forms. The presence of these NAF forms as variants of M. tuberculosis, and not merely contaminants, was clearly established by a number of methods. These included phase contrast and electron microscopy, immunological studies employing antiserum and comparison with the parent organisms, and gel electrophoresis of the proteins of the parent organisms. Other evidence is also offered confirming the hypothesis of reversion of NAF forms. It is also shown in this study that NAF forms can be induced in vitro by hydrolases.


Tuberculosis and HIV infection association in a rural district of northern Uganda: epidemiological and clinical considerations.

59 (18.3%) of 323 patients with tuberculosis (TB) tested for HIV-1 antibody by ELISA technique (Wellcozyme) were seropositive. In the control group selected among the health personnel working in the Arua Hospital, 7.7% were found positive for HIV-1 antibody, thus showing a significantly lower prevalence compared with the TB patients (P < 0.005). The prevalence of HIV infection was 50% in the urban TB patients, 7% in TB patients living in rural areas surrounding Arua town and 1.6% in the peripheral rural setting. Of 27 TB patients with clinical AIDS, 18 died during the course of the study. The AIDS patients' survival rate was 46.4% 6 months after diagnosis, and 21.4% after 16 months, the median period of survival being 5.0 months. Risk factors, sputum conversion rate, clinical and radiological findings were analysed. No significant difference was found between seropositive and seronegative TB patients for clinical drug-related toxicity (P > 0.05).


Effect of birth season on case fatality rate of pulmonary tuberculosis: coincidence or causality?

The effects of birth season on the case fatality rate (CFR) of pulmonary tuberculosis were examined in case records of 9206 patients discharged during 1937-1965 from a national hospital for tuberculosis in Tokyo. The CFR was over 60% until 1945, then it decreased down to less than 9% after 1950. Male cases had little variation in their CFR by their birth season, while female cases had a significant variation and showed a significantly higher CFR than the male cases when they were born in January-June. The results suggest that the birth season had a significant influence on the prognosis of pulmonary tuberculosis.


The supply of antituberculosis drugs and national drugs policies.

The money necessary for purchasing antituberculosis drugs in a national programme comes from the Public Health Services budget, funds from the health insurance scheme (when it exists) and household budgets. The Public Health Services budget is on average $2-23 US per year and per person in low or middle income countries. Average drug consumption in these groups of countries was from $2-26 US per person in 1990. The number of patients to be treated depends on the annual risk of tuberculous infection (ARTI) and the case detection rate: it is the same when the ARTI is 1% and the detection rate 100%, and when the ARTI is 2% and the detection rate 50% of cases. The supply of antituberculosis drugs has a real cost representing around 3% of global drug consumption in low income countries and is always less than 1% of global drug consumption in middle income countries. In most developing countries it could be covered with the aid of national financial resources, on condition that it is integrated into a coherent national drugs policy aimed at guaranteeing the continued availability of essential drugs.


Secretory functions of the vascular endothelium.

The endothelial cells which line the blood vessels as a monolayer exert a remarkable control over the vascular system. Indeed, the endothelium can be regarded as a highly active metabolic and endocrine organ in its own right. On the hand, vasoactive substances such as serotonin and bradykinin are inactivated and on the other the cells can enzymatically produce the vasoconstrictor, angiotensin II and secrete endothelin-1 ((ET-1). Perhaps more importantly, the cells also produce two unstable vasodilator substances, which potently inhibit platelet clumping: prostacyclin and endothelium-derived relaxing factor (EDRF) which has been identified as nitric oxide (NO; 1). Both substances seem well designated as local hormones, released to influence adjacent cells. The endothelial cell, therefore, exerts control over the cardiovascular system by elaborating dilator substances as well as vasconstrictors.


Regional heterogeneity of myocardial blood flow within the rabbit left ventricle during haemorrhagic hypotension.

Several studies have reported an extensive regional heterogeneity in myocardial blood flow. The reported coefficients of variation for regional myocardial perfusion range from about 0.2 to 0.4 in normotensive animals. The spatial distribution of myocardial perfusion during haemorrhagic hypotension seems not to have been assessed. The goal of the present study was to determine the regional heterogeneity in myocardial blood flow within the rabbit left ventricle during normal conditions and after haemorrhagic hypotension. Radioactive microspheres were infused into the left ventricle in barbiturate anaesthetized rabbits over either 30 or 120 sec. The haemorrhagic hypotension was induced by bleeding, so that mean arterial blood pressure was reduced to about 50% of control. The left ventricles were divided into samples of about 0.025 g each. Regional heterogeneity in the blood flow was expressed as the coefficient of variation corrected for the Poisson distribution of microspheres (CVc). The CVc was 0.37 +/- 0.09 (mean +/- SD) during control and 0.41 +/- 0.11 after bleeding, the CVc obtained after bleeding being somewhat higher than during control (P < 0.05). We obtained a high correlation coefficient (tau about 0.68) between regional perfusion values at control and after bleeding which indicates a stable perfusion pattern within the myocardium. We conclude that the regional distribution of coronary blood flow within the left ventricle is markedly heterogenous during control condition and that this pattern is not changed during haemorrhagic hypotension.


Effect of exercise on adenosine deaminase activity in rat skeletal muscles.

Adenosine deaminase activity was shown to decrease in each skeletal muscle type (the slow-twitch oxydative, fast-twitch oxydative--glycolytic and fast-twitch glycolytic) at the beginning of exercise of moderate intensity and to return to the control when exercise was continued till exhaustion. 5 min occlusion of the femoral artery had no effect on the enzyme activity in either muscle. The reduction of the enzyme activity at the onset of exercise could result in reduction of adenosine breakdown and thus contribute to vasodilation at this stage of increased contractile activity of the muscles.


Feedback control of pancreatic secretion in rats. Role of gastric acid secretion.

Pancreatic secretion in rats is regulated by feedback inhibition of cholecystokinin (CCK) release by proteases in the gut lumen, but little is known about the role of gastric acid in this regulation. This study, carried out on conscious rats with large gastric fistulas (GF) and pancreatic fistulas, shows that diversion of pancreatic juice results in the progressive stimulation of pancreatic secretion only in rats with the GF closed. When the GF was kept open, the diversion resulted in only small increment in pancreatic secretion and this was accompanied by progressive increase in gastric acid outputs. Similar amounts of HCl instilled into the duodenum in rats with the GF open fully reproduced the increase in pancreatic secretion observed after the diversion of pancreatic juice. Pretreatment with omeprazole (15 mumol/kg) to suppress gastric acid secretion or with L-364,718 (5 mumol/kg) to antagonize CCK receptors in the diverted state, resulted in the decline in pancreatic secretion similar to that observed after opening the GF. CCK given s.c. (20-320 pmol/kg) failed to cause any significant rise in the post-diversion pancreatic secretion in rats with the GF closed, but stimulated this secretion dose-dependently when the GF was open. Camostate (6-200 mg/kg) in rats with pancreatic juice returned to the duodenum caused dose-dependent increase in pancreatic secretion, but after opening the GF or after omeprazole this increase was reduced by about 75%. This study provides evidence that gastric acid plays a crucial role in the pancreatic response to diversion of pancreatic juice or inhibition of luminal proteases, and that factors that eliminate gastric acid secretion reduce this response.


Effect of gamma-aminobutyric acid and muscimol on corticosterone secretion in rats.

The effect of gamma-aminobutyric acid-receptor agonists, GABA and muscimol on the pituitary-adrenocortical activity, measured indirectly through corticosterone secretion, and the receptors involved were investigated in conscious rats. GABA given ip induced a dual effect, in lower dose (10 mg/kg) it significantly decreased the resting serum corticosterone levels while in higher doses (100-500 mg/kg) it considerably raised that level. Muscimol (0.5 mg/kg ip) also increased the corticosterone concentration. Both GABA and muscimol given intracerebroventricularly (icv) induced a significant, dose-related increase in serum corticosterone levels. Bicuculline, a GABAA-receptor antagonist, totally abolished the corticosterone response to GABA but did not influence the response to muscimol. Pretreatment with atropine did not affect the corticosterone response to GABA but significantly diminished the response to muscimol. These results suggest that GABA moderately inhibits the pituitary-adrenal axis at the pituitary level but significantly stimulates it at the hypothalamic level. The stimulatory effect of GABA, but not muscimol, is mediated by hypothalamic GABAA-receptors, and in the effect of muscimol hypothalamic cholinergic, muscarinic receptors are involved to a significant extent.


Behavioural effects of luteinizing hormone-releasing hormone (LHRH) in rats.

Behavioural effects of intracerebroventricularly-injected (icv) LHRH were studied in female rats. Locomotor and exploratory activities as well as irritability were determined. A pronounced inhibitory effect of 10 micrograms doses of LHRH was found. At 100 micrograms doses of LHRH, barrel behaviour was observed. We conclude that LHRH can modify the activity of central serotonergic receptors in rats.


The influence of age and sex on selenium distribution and glutathione peroxidase activity in plasma and erythrocytes of selenium-adequate and supplemented rats.

The experiment was performed on Sprague-Dawley male rats weighting 203, 103 and 53 g, and female 99 g. Animals were fed for 2 weeks a diet containing 0.1 and 2.0 ppm of Se (Na2SeO3 added). It was observed that the daily Se intake per kg of BW is lowered with an increase in animals body weight. Se-supplementation caused a significant increase of Se content in plasma and red blood cells. The highest concentration of Se in plasma and in RBC was found in females. GSH-Px activity was higher in RBC of all male rats receiving a Se-supplemented diet, but not in females. In plasma these differences between Se-adequate and supplemented rats were significant in youngest male rats and in females. These results suggest that age and sex of rats affect the concentration of Se and GSH-Px activity in plasma and RBC of rats.


[Pathology of stomach cancer].

Dysplasias, the best-defined precancerous lesion of the stomach, are seldomly found alone, but most commonly in association with carcinoma. The established classification of gastric carcinomas includes the site, histological tumour type, grade of differentiation, R classification and pTNM classification with stage grouping. The Laurén classification is important in operative planning (extent of operation on the stomach and oesophagus). Additional conventional classification parameters are at present not proven independent prognostic factors.


[Surgical treatment of early stomach cancer].

Data from our own clinical experience as well as overview articles from Japan show an 80%-100% 5-year survival rate for early gastric cancer. An indispensable prerequisite is a resection depending on the type, size and infiltration. For the submucosal type, which shows lymph node metastasis in 10%-25%, the appropriate surgical therapy is resection of the stomach or total gastrectomy with systemic lymphadenectomy of compartments I and II. Distinct types of mucosal carcinoma (small size) may be suitable for local therapy, particularly in high-risk patients.


[Indications for aboral subtotal resection].

Aboral gastric resections for gastric cancer are performed in 20%-40% of cases at German university hospitals. This type of resection can only be carried out if an ample oral safety margin is given. The margin should be at least 10 cm in the case of diffuse carcinoma, and no less than 6 cm in intestinal-type carcinoma. Thus, aboral subtotal resection is confined to smaller tumors, preferably those located in the antrum of the stomach. Lymph node dissection can and should be carried out in the case of subtotal resection. Aboral subtotal resection represents an adequate form of treatment, complying with the necessary oncological standards if the appropriate safety margin is chosen.


Wert der systematischen erweiterten Lymphknotendissektion--Ergebnisse in Japan. [Value of systematic extended lymph node dissection--results in Japan].

In the National Cancer Center, 6112 patients with primary gastric cancer have undergone gastric resection since 1962, and the 5-year survival rate (5YSR) was 57.5%. In the 25-year period, 5YSR rose from 57% to 83% in stage II disease, and from 33% to 50% in stage III disease. Systematic lymph node dissection (R2 dissection) played major role in this improvement. It gave significantly better prognosis (5YSR, 48%) than incomplete dissection (31%), particularly reducing local recurrence. The proportion of local recurrence was 38% in 1967-1971, however it decreased to 16% in 1982-1986 by introduction of this treatment. Disadvantages of the dissection were minimal. Surgical death rate was 3.0% in the 1960s but it was only 0.6% in the 1980s.


[Left upper abdominal quadrant evisceration].

To improve the surgical outcome of patients with advanced gastric cancer, we performed left upper abdominal quadrant evisceration (LUAE), which was introduced by Prof. T. Kajitani in 1980. From 1980 to 1989, 0.2% of 5123 gastric resections performed in the cancer institute Hospital and the National Cancer Center were total gastrectomies, in 4.5% (i.e., 232 patients) LUAE was the operative treatment. The 5-year survival rate of patients who underwent LUAE was 28%. Complications of the operation were 39.6%, other complications occurred in 15.1%. The mortality of patients who underwent LUAE was 2.2%.


[Stomach cancer: multimodality therapy--reliable and new developments].

The extension of surgical radicality including multivisceral procedures has only improved prognosis in individual cases. The concern now obviously is to achieve an improvement by additional therapeutic measures. Unfortunately, studies in Western countries show that adjuvant chemotherapy does not result in any significant improvement as has been repeatedly reported in Japanese trials. It is possible that differences in time and mode of drug application may be responsible for these discrepancies. Decisive may be the immediate post- or intraoperative therapy as well as its intraperitoneal application. Some phase II trials have also shown that preoperative chemotherapy may improve the resectability rate. Further studies are needed to determine how these findings are generally transferable. In addition of chemotherapy intraoperative radiation (IORT) and photodynamic therapy (PDT) may gain importance in the treatment of gastric cancer for the future.


[Mesothelioma of the pleura--pathological anatomy].

The examination of the pathology of diffuse malignant mesotheliomas (DMM) is based upon the analysis of 600 pleural tumours of the German Mesothelioma Panel in Bochum. Macroscopically there is a characteristic feature of the tumour growth, often restricted to the pleura, with preference of the basal pleural parts even in advanced stages. The highly variable growth pattern of the tumour phenotype contains parts of epithelial cells (42%), sarcoma-like cells (18%), and biphasic patterns (27%) as revealed by microscopy. Secondary pleural carcinosis are much more frequent (10 x) as diffuse malignant mesotheliomas but may mimic the DMM. Since there is no specific tumour marker for DMM available, additional immunohistochemical staining using different monoclonal antibodies directed against cytokeratins and epithelial antigens are recommended especially for examinations of small biopsies. The morphological security cheque follows the criteria of the European Mesothelioma Panel and is subdivided into 5 groups: A, assured mesothelioma; B, possible mesothelioma; C, probable mesothelioma; D, possibly no mesothelioma; E, certainly no mesothelioma. In the Bochum panel the incidence of A and B is 66%, of C, 6% and of D and E, 28%. In addition to the histomorphological examinations, in dust analysis of nontumourous lung tissue an augmented asbestos charge has been found in approximately 90% of all cases. The significance of asbestos fibers as an essential cause of the pleural mesothelioma could be underlined by our own animal experiments. After instillation of asbestos fibers into the right lung, a transpleural transport of fibers was observed with preneoplastic lesions and transformation of multipotent subserosal cells.


[Mesothelioma of the pleura--diagnosis with imaging procedures].

Typical radiological findings in patients with diffuse malignant pleural mesothelioma are nodular opacities of the pleura including the fissures, pleural effusion, shrinking of the ipsilateral hemithorax, and mediastinal shift. The radiological differentiation between pleural mesothelioma and benign pleural tumors is possible; however, a secondary metastatic pleural involvement imitates pleural mesothelioma. Magnetic resonance imaging is superior to computed tomography in the preoperative assessment of tumor extension, because it demonstrates exactly the tumor extension in the coronal or sagittal plane. The noninvasive ultrasound examination is helpful in the evaluation of the diaphragmatic pleura and the costodiaphragmatic spaces.


[Progress in heart transplantation].

Within the past 10 years, heart transplantation has become established as a standard procedure in heart surgery. Improvements in immunosuppressive therapy and diagnosis of graft rejection have been crucial. The criteria for transplantation have been broadened for recipients as well as for donors. Newborns, pediatric patients, diabetics, and patients with impaired renal function will no longer be excluded from transplantation due to improved postoperative therapy. Furthermore, progress has been made with assisted circulation. Patients with acute heart failure can now be bridged to transplantation.


[Progress in the field of liver transplantation in the last 10 years].

During the past 10 years, liver transplantation has developed into an established form of treatment. In 1991 alone, almost 450 liver transplants were performed in Germany with a mean 1-year survival of 70%. Consistent progress in the fields of organ preservation, surgical techniques, and prevention of disease recurrence has made a more than 90% 1-year survival rate achievable during the last 3 years in Berlin. Progress in organ preservation is based on the use of colloidal solutions, pharmacological improvement of microcirculation, and probably prevention of reperfusion injury by the use of calcium antagonists and free oxygen radical scavengers. Improvements in surgical techniques have led to a reduction in blood loss and vascular complications, and to a drastic reduction of biliary leaks and stenosis. The introduction of cyclosporin, FK-506, and monoclonal antibodies into immunosuppressive therapy has contributed to the improvement of results in a major way. Furthermore, progress in intensive care in the areas of fluid therapy and blood component substitution, respiratory care and prophylaxis of infection as well as postoperative nutrition has led to a reduction in early postoperative morbidity and mortality. Efficient prophylaxis of the recurrence of hepatitis-B-virus-related disease by using hepatitis B hyperimmunoglobulin after liver transplantation has prevented this lethal recurrence, which was previously frequent, in a large group of patients.


[Progress in kidney transplantation].

The introduction of immunosuppression with cyclosporine A in 1983 compared to conventional immunosuppression has clearly improved early results within the first year post transplant. In contrast, the rate of graft failure per year in the following course has not changed essentially. Still HLA histocompatibility is a predominant prognostic factor which can be influenced by organ sharing. On the other hand, donor factors like sex and age are becoming more important and should be taken into account for organ allocation. This furthermore requires strict attention to future exchange rules within the transplant community.


[Development and importance of combined organ transplantation].

In 1966, Kelly and Lillehei transplanted a pancreas together with a kidney in a young patient suffering from end-stage diabetic nephropathy. Since this first combined organ transplantation, various organs have been transplanted together: heart-lung (Cooley 1969), liver-kidney (Margreiter 1983), liver-pancreas-stomach-small bowel (Starzl 1983), heart-liver (Starzl, Bahnson 1984), heart-lung-liver (Wallwork, Calne 1986), and liver-small bowel (Grant 1990). Although some of these combined organ transplants will only occasionally be necessary, some others such as kidney-pancreas, heart-lung, or liver-kidney have already gained clinical relevance.


[Developments in the field of transplantation of bones and cartilage].

Experience with more than 2000 allogenic bone and cartilage transplantations between 1975 and 1990 confirms the indication for implanting this material in a wide range of applications. Especially in reconstructive surgery and after tumor resection the use of this procedure is increasing. The main advantage is the almost unlimited availability and the possibility of en bloc and orthotopic administration. Even with extensive defects, we saw satisfying results. An overall complication rate of 4.5% is low, and infection is the complication most frequently seen. Primary consolidation was seen in over 80% on average after 17 weeks.


[Organ transplantation, development and perspectives of the legal status].

The FRG has no transplantation law as most other European countries do. A model of agreement is practised which is derived from fundamental principles, i.e., a personal right outlasting death and the agreement of the next of kin for organ removal. The paper favours a legal regulation that resolves the difference between a model of presumed consent on the hand and a model of agreement on the other hand, and advocates informing the next of kin, who can then object as representatives of the deceased.


[Organ donation to hospitals not specializing in transplantation].

The necessary and possible rate of transplantations is limited by the insufficient availability of donor organs. In the FRG, only 36% of all hospitals (transplantation centers excluded) are active in postmortem organ donation. In only 57% are other organs than the kidneys (liver, pancreas, heart, lung) explanted. The main problems do not derive primarily from structural difficulties (no intensive care unit, limited capacity of operation room or staff), but from an insufficient degree of cooperation with the transplantation center and an uncertain legal position. Possible solutions to the problem (transplantation coordinator, legal regulation) are discussed.