Panic, suicide, and agitation: independent correlates of the TSH response to TRH in depression.

We investigated the relationship between suicidality, agitation, panic attacks, and the thyrotropin-stimulating hormone (TSH) response to thyrotropin-releasing hormone (TRH), and tested the hypothesis that panic would account for the association between a reduced TSH response and the other conditions. Twenty-seven euthyroid primary unipolar depressed inpatient women received a TRH test and systematic psychiatric assessment. Panic attacks were insufficient to explain the link between the TSH response and suicidal intent, lethality, and agitation; each condition was independently associated with a lower TSH response. In an additive fashion, copresence of conditions further reduced TSH response. The symptom constellation of panic, agitation, and suicidality in depression may correlate with the greatest reduction in TSH response.


Chaotic dynamics in autonomic nervous system activity of a patient during a psychotherapy session.

Chaos theory and nonlinear dynamical modeling have been applied successfully in a variety of scientific fields ranging from the physical sciences to population biology. More recently, investigators have modeled long-term psychological processes by applying this paradigm to subjective measures. Yet, mathematically rigorous dynamical modeling of transient psychodynamic processes remains uncharted. Human behaviors and intrapsychic processes often defy prediction, yet psychodynamic theory views these as fundamentally deterministic. We postulate that a nonlinear dynamical analysis of mental states and mental control processes may help reconcile the paradox that seemingly random mental states are thought ultimately to result from deterministic, rather than stochastic, psychological processes. As an initial empirical approach, we examined spontaneously occurring autonomic activity of a patient during 1 hr of psychotherapy and illustrate that cardiac responses associated with psychologically meaningful events possess nonlinear characteristics indicative of chaos. Such nonlinear analyses may shed light on fundamental issues of psychological functioning.


Effects of human chorionic gonadotropin and progesterone administration on porphyrin biosynthesis and histology of the Harderian glands in male and female Syrian hamsters.

We investigated the influence of hCG and progesterone on the control of porphyrin biosynthesis and histology in the Syrian hamster Harderian glands. Castration of male hamsters caused a marked elevation in porphyrin biosynthesis as revealed by the concentrations of porphyrins and the mRNA levels of the porphyrin pathway rate-limiting enzyme, 5-aminolevulinate synthase (ALV-S). Injection of hCG into castrated male hamsters also resulted in a significant increase in both porphyrin concentrations and levels of ALV-S mRNA compared with those in saline-injected castrated hamsters. Type II cells, which are filled with large lipid vacuoles and are characteristic of male phenotype, disappeared after castration, but administration of hCG partially prevented this change. On the other hand, neither administration of hCG nor progesterone implants could increase the very high porphyrin concentrations and ALV-S mRNA levels characteristic of female Syrian hamsters. As in the case of castrated male hamsters, injections of 20 IU hCG to female Syrian hamsters increased the relative number of Type II cells per square millimeter, whereas progesterone administration did not modify the relative number of Type II cells. These results indicate that hCG can modify Harderian gland morphology in both male and female hamsters and can exert a positive control in the expression of ALV-S gene in castrated male hamsters.


Localization and synthesis of entactin in seminiferous tubules of the mouse.

Localization and synthesis of entactin in seminiferous tubules of mouse testis was studied by immunocytochemistry. Frozen sections from adult mice testes were subjected to anti-entactin and anti-laminin immunofluorescence. Both entactin and laminin were localized within the seminiferous tubule basement membrane and intertubular region of the testis. The addition of excess amount of entactin (but not fibronectin), premixed with anti-entactin antiserum, abolished the immunostain. Western blotting showed that a protein extract from a seminiferous tubule basement membrane preparation was recognized by anti-entactin anti-serum and comigrated with recombinant entactin. Enriched fractions of isolated primary Sertoli cells and peritubular myoid cells cultured for 6 days on a glass coverslip were able to synthesize and secrete entactin as detected by immunofluorescence microscopy. Entactin was also produced by TM3 (Leydig-like) and TM4 (Sertoli-like) cell lines as detected by both immunofluorescence and Western blotting. The distribution of entactin vs. laminin within both the cultured primary cells and the TM3 and TM4 cell lines differed. Entactin appeared mainly localized extracellularly. In contrast, laminin was mainly localized intracellularly. The above findings suggested that 1) entactin existed in the seminiferous tubule basement membrane and intertubular region of adult mice testis, co-localized with laminin; 2) entactin was synthesized by the cultured primary Sertoli cells and peritubular myoid cells and the TM3 and TM4 cell lines; 3) entactin was exocytosed with little intracellular accumulation, in contrast to an intracellular accumulation of laminin.


Synthesis of uterine endometrial proteins during early diestrus in the cyclic and pregnant dog, and after estrogen and progesterone treatment.

The objectives of this study were to identify and characterize dog uterine endometrial proteins synthesized de novo in explant culture during early luteal phase, to examine distribution of these proteins prior to the embryo's entering the uterus and during its free-floating period prior to implantation, and to examine regulation of endometrial proteins by estrogen and progesterone (P4) treatments. Uterine endometrium was collected from cyclic and pregnant bitches on diestrus Days 3, 7, and 10 as determined by loss of cornification of vaginal epithelium, and from ovariectomized dogs after treatment with corn oil, estrogen, P4, or estrogen followed by 1 or 2 wk of P4. Tissue was incubated in an explant culture system in the presence of [3H]leucine or [35S]methionine. The rate of incorporation of [3H]leucine into nondialyzable macromolecules indicated no significant change in rates of incorporation by status (pregnant vs. nonpregnant), day, or steroid treatment. Uterine endometrial-conditioned culture medium, analyzed by two-dimensional SDS-PAGE and fluorography, revealed a complex array of at least ten proteins or protein complexes in cyclic and pregnant bitches. No difference in protein pattern was detected by status; however, differences in distribution were apparent by day of cycle or early pregnancy. Two major proteins, cP5 (M(r) 54,686) and cP6 (M(r) 23,010) appeared to be differentially expressed. Expression of cP5, maximal on diestrus Day 3, decreased as the cycle or pregnancy progressed to diestrus Day 10. In contrast, expression of cP6, a minor protein on diestrus Day 3, appeared to be up-regulated for each status to Day 10, with increased intensity and multiple isoelectric and molecular-weight variants. In ovariectomized steroid-treated dogs, two-dimensional SDS-PAGE showed that pattern and distribution of specific proteins were affected by treatment. Acidic protein cP1 (M(r) 87,600), synthesized after corn oil and P4 treatment, was suppressed with estradiol (E2). Proteins cP2 (M(r) 40,000 and M(r) 42,000), present with all treatments, were intensified with P4. A high-M(r) basic protein complex (cP3) and acidic protein cP4 were expressed with E2 and maintained with P4 treatment. Proteins cP5 and cP6, while not induced by E2 or P4 alone, required E2 priming for P4 induction. Protein cP5 was down-regulated while cP6 was up-regulated with P4 for 2 wk. Proteins induced by estrogen followed by 1 or 2 wk of P4 treatments were similar to those released by endometrial explants collected from pregnant and cyclic bitches on Days 3, 7, and 10 of spontaneous diestrus.(ABSTRACT TRUNCATED AT 400 WORDS)


Immunological identification of G protein alpha- and beta-subunits in tail membranes of bovine spermatozoa.

Heterotrimeric G proteins are believed to play important roles as signal transducing components in various mammalian sperm functions. To assess the distribution of G proteins in bovine sperm tails, we purified membranes by hypoosmotic swelling of bovine spermatozoa followed by disruption of plasma membranes in a homogenizer and various centrifugation steps. Electron microscopy revealed highly purified membranes of bovine sperm tails. Subsequently, antisera against synthetic peptides were used to identify G proteins in immunoblots. An antiserum directed against the C-terminal decapeptide of Gi3 and detecting all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. In contrast, various other specific peptide antisera against alpha-subunits did not detect any G protein in enriched tail membranes. An antiserum recognizing the beta 2-subunit of G proteins and an antiserum reacting with both beta 1- and beta 2-subunits identified a 35-kDa protein in sperm tail membranes. In contrast, antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. Our results suggest that G proteins in membranes of tails of bovine spermatozoa most likely belong to a novel subtype of G protein alpha-subunits, whereas the putative beta-subunit could be identified as a beta 2-subunit.


Production of chimeric hamsters by aggregation of eight-cell embryos.

Chimeric animals were produced by aggregation of 8-cell-stage embryos from two strains of hamsters (LVG and Bio 1.5). Two series of experiments were performed. In the first series, embryo pairs in contact with each other were classified as aggregates even if 2 distinct embryos could still be distinguished. Of 88 aggregates transferred, 2 chimeras were obtained. Pregnancy rate was 25%, and embryo survival was 35%. In the second set of experiments, only embryo pairs that had coalesced to form a single giant blastocyst were classified as aggregates. Of 56 aggregates transferred, 6 chimeras were obtained. Pregnancy rate was 83%, and embryo survival was 30%. Of the 8 chimeras, 6 were phenotypic males, and 2 were phenotypic females. Both females were germ line chimeras. Of the 6 males, 4 reproduced normally, 1 had abnormal external genitalia but normal spermatogenesis, and 1 was sterile and had atrophic testes. Each of the fertile males transmitted only a single component, either the LVG or the Bio 1.5. Examination of the testes from the sterile chimera revealed that in excess of 80% of the seminiferous cords were devoid of germ cells. These results demonstrate that hamster chimeras can be obtained by aggregation of 8-cell-stage embryos.


Gonadotropin surge increases fluorescent-tagged low-density lipoprotein uptake by macaque granulosa cells from preovulatory follicles.

In the primate ovary, luteal steroidogenesis is largely dependent upon cholesterol derived from receptor-mediated uptake of circulating low-density lipoprotein (LDL). However, granulosa cells (GC) of preovulatory follicles possess few LDL binding sites compared to those present in developing and mature corpora lutea. We recently reported (Endocrinology 1991; 129:3247-3253) that uptake of LDL tagged with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) can be monitored in macaque luteal cells by fluorescence-activated flow cytometry. This study was designed to determine whether an ovulatory stimulus induced uptake of DiI-LDL in GC aspirated from preovulatory follicles of macaques undergoing ovarian stimulation. Development of multiple large follicles was stimulated in adult rhesus macaques with human gonadotropin treatment for 9 days. On Day 10, monkeys received either no ovulatory stimulus or 1000 IU hCG to initiate ovulatory events. GC were aspirated on Day 10 in monkeys receiving no ovulatory stimulus (nonluteinized GC) or 27 h or 34 h after hCG injection (luteinizing GC). GC were resuspended in Ham's F-10 medium + 0.1% BSA and incubated with several concentrations (0-25 micrograms/ml) of DiI-LDL (Biomedical Technologies, Stoughton, MA) for various time intervals (2-60 min). DiI-LDL uptake by GC was time- and concentration-dependent. Coincubation of cells with DiI-LDL and unlabeled LDL dose-dependently suppressed the percentage of fluorescent cells. In contrast, coincubation with up to a 250-fold excess of acetylated LDL or high-density lipoprotein did not alter the percentage of fluorescent GC.(ABSTRACT TRUNCATED AT 250 WORDS)


Active and passive immunoneutralization of inhibin increases follicle-stimulating hormone levels and ovulation rate in ewes.

Two experiments were conducted to determine effects of active and passive immunoneutralization of inhibin on FSH secretion and ovulation rate. A synthetic peptide (alpha-IF) matching the N-terminus of the alpha-subunit of ovine inhibin was coupled to human alpha-globulin (h alpha-G) and used as an immunogen. In experiment 1, estrus was synchronized in 10 sheep that had been actively immunized against alpha-IF-h alpha-G or h alpha-G. Plasma FSH levels were similar in the two groups of ewes at -52 and -48 h (0 h = onset of estrus). In alpha-IF-h alpha-G-immunized ewes, FSH increased from -48 to -44 h (18.8-22.1 ng/ml), and then fell to 16.2 ng/ml by 0 h. In h alpha-G-immunized ewes, FSH decreased from -48 to 0 h (17.6-7.2 ng/ml). Ovulation rate was higher in alpha-IF-h alpha-G- than h alpha-G-immunized ewes (9.4 vs. 2.4). In experiment 2, antibodies (Ab) were extracted from sera obtained from experiment 1 ewes and then were injected i.v. into 12 other ewes. Estrus was synchronized twice during the breeding season using progesterone-releasing pessaries (CIDR-G). One day before CIDR-G withdrawal, alpha-IF-h alpha-G and h alpha-G Ab were administered in a crossover design. After injection of Ab against alpha-IF-h alpha-G, plasma FSH increased from 0 to 24 h post-injection (10.9-21.5 ng/ml), after which levels fell to 14.2 ng/ml by onset of the preovulatory LH surge.(ABSTRACT TRUNCATED AT 250 WORDS)


Decreased luteinizing hormone-stimulated progesterone secretion by preovulatory follicles isolated from cyclic rats treated with the progesterone antagonist RU486.

Since administration of the antiprogesterone RU486 to cyclic female rats at metestrus and diestrus results in increased serum levels of LH, estradiol, and testosterone at proestrus, we investigated whether RU486 affects follicular steroidogenesis. Female rats with a 4-day estrous cycle, induced experimentally by a single injection of bromocriptine on the morning of estrus, were given RU486 (2 mg) twice daily (0900 and 1700 h) on metestrus and diestrus. At proestrus the preovulatory follicles were isolated and incubated for 4 h in the absence and presence of LH. In the absence of LH, accumulation of estradiol, testosterone, and progesterone in the medium was not different for RU486-treated rats and oil-treated controls. In contrast, LH-stimulated estradiol, testosterone, and progesterone secretions were significantly lower in RU486-treated rats compared with controls. Addition of pregnenolone to the incubation medium resulted in a significantly lower increase of progesterone in follicles from RU486-treated rats compared with those from oil-treated controls. This suggests that 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity is decreased by administration of RU486 in vivo. Aromatase and 17 alpha-hydroxylase/C17-20 lyase activities were not affected: addition of substrate (androstenedione and progesterone respectively) did not affect differently the amount of product formed (estradiol and testosterone) in RU486- and oil-treated rats. However, LH-stimulated pregnenolone secretion was lower in follicles from RU486-treated rats compared with follicles from oil-treated controls, suggesting that either cholesterol side-chain cleavage activity or LH responsiveness is decreased. At proestrus the preovulatory follicles from RU486- and oil-treated rats were not morphologically different.(ABSTRACT TRUNCATED AT 250 WORDS)


Expression of bovine trophoblast interferon in conceptuses derived by in vitro techniques.

Expression of the trophoblast interferon, bovine trophoblast protein-1 (bTP-1), has been studied in embryos produced by in vitro maturation-in vitro fertilization (IVM-IVF). No bTP-1 production was noted until after embryos had reached the expanded blastocyst stage and had begun to hatch (Days 8-9 post-fertilization). Single blastocysts comprising 115 +/- 22 cells released 1.0 +/- 0.1 units of interferon activity/24 h. Amplification of conceptus mRNA by reverse transcription-polymerase chain reaction procedure with bTP-1-specific oligonucleotides confirmed that bTP-1 transcripts were present in blastocysts but were not detectable at earlier stages. Although cultured blastocysts produced by IVM-IVF procedures continued to secrete bTP-1 for a few days, they failed to attach to the substratum and form outgrowths, and soon lost structural integrity. However, when Day 8 blastocysts/morulae were transferred to the uteri of synchronized cows, recovered 4 days later, and placed into individual cultures, they attached and formed outgrowths that produced large amounts of bTP-1 (greater than 2000 units/culture/24 h after 14 days). Embryos thus first expressed bTP-1 when a functional trophectoderm was first formed, and induction did not require a period of in vivo development. However, continued viability of the blastocyst and bTP-1 production were not sustained in vitro and may require some exposure to the uterine environment.


Beta-nerve growth factor influences the expression of androgen-binding protein messenger ribonucleic acid in the rat testis.

The effect of infusion of nerve growth factor (NGF) into the rat testis on the expression of androgen-binding protein (ABP) mRNA was studied. A major 1.7-kb and a minor 3.7-kb ABP mRNA were present at all stages of the seminiferous epithelium with maximal levels at stages VIII-XI and the lowest levels at stages IV-VI. Infusion of 15 ng/h of NGF with a mini-osmotic pump for 14 days resulted in a 2-fold increase of ABP mRNA as revealed by Northern blots, whereas the mRNA level of another Sertoli cell protein, urokinase-type plasminogen activator, remained unchanged. Image analysis of autoradiograms obtained by in situ hybridization of sections from treated testes showed a similar increase in APB mRNA compared to noninfused or PBS-infused testes. However, at the cellular level the labeling intensity for ABP mRNA over Sertoli cells of different stages of the seminiferous epithelium was the same in NGF-infused and control testes. This suggests that the increase of ABP mRNA in NGF-infused testes was caused by prolongation of stages VII-VIII with maximal ABP mRNA expression; the suggestion is supported by an increase of 30 percent in frequency of these stages in histological sections from NGF-infused testes.


Prenatal androgens and the timing of seasonal reproductive transitions in sheep.

Both the onset of puberty in the lamb and the annual resumption of reproductive activity in adult male and female sheep are characterized by increased secretion of LH due to reduced responsiveness to steroid inhibition. However, the timing of puberty is sexually differentiated, for males undergo a reduction in sensitivity to steroid feedback at 10 wk of age, whereas females remain highly responsive to steroid inhibition until 30 wk. This sex difference is determined by androgens in utero. The present study was conducted to determine whether a sex difference exists in the timing of seasonal transitions in adult males and females. We compared serum LH in gonadectomized, estradiol-treated males (n = 7), females (n = 6), and androgenized females (n = 5) from blood samples collected twice weekly for one year. As determined by changes in the pattern of LH secretion, the onset and termination of the autumn breeding season were not different between males, females, and androgenized females (termination: 1 February +/- 4 days, mean +/- SE all groups; onset: males, 22 August +/- 4 days; females, 5 September +/- 18 days; androgenized females, 16 September +/- 10.5 days). However, there was a transient increase in LH (20 May to 23 June) in males, but not in females or androgenized females. Although no effects of prenatal testosterone were evident in the control of LH secretion in adult androgenized females, LH secretion in androgenized males was elevated throughout the nonbreeding season in 3 of 5 animals, indicating that exogenous testosterone may reduce seasonal increases in responsiveness to steroid inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)


The effect of estrogen and progesterone on structural changes in the uterine glandular epithelium of the ovariectomized sheep.

The development of epithelial cells of the uterine glands of ovariectomized sheep in response to estradiol-17 beta (E) and progesterone (P) was studied using light and electron microscopy. Animals that had been ovariectomized for six weeks were placed in one of three experimental treatment groups. Group I animals (untreated controls) received no steroid treatment. Group II animals (E alone) received one 4-cm E implant (E approximately 5-10 pg/ml) and their uteri were removed after 2, 4, or 6 days. Group III animals (E-primed, P-treated) received an E implant (E approximately 5-10 pg/ml) for 6 days and then were treated with six 13-cm P implants (P approximately 1.5-3 ng/ml), in the continued presence of E, for 2, 4 or 6 days. Six weeks after ovariectomy the epithelial cells of the uterine glands were low cuboidal and morphologically appeared to be synthetically inactive. Following 2 days of E treatment the epithelial cells had significantly increased in cell height, and protein-synthesizing organelles were well developed. Maturation of the secretory apparatus continued throughout E treatment. The Golgi complex and rough endoplasmic reticulum (RER) were abundant. Lysosomal-like granules and granules of varying electron density were present in the cytoplasm. The chronic administration of P to E-primed animals did not result in any further increase in cell height. Elongated mitochondria, a cup-shaped Golgi apparatus, extensive apical microvilli, and irregularly shaped membranous profiles in the supranuclear cytoplasm characterized these uterine epithelial cells. Lysosomal-like granules, small vesicles, and scattered patches of glycogen were seen in the cytoplasm. These data show that the uterine epithelial cells of the ovariectomized sheep undergo morphological alterations in protein-synthesizing organelles and apical specializations that depend on the presence of E and P.


A two-receptor model for salmon gonadotropins (GTH I and GTH II).

The possible existence of distinct receptors for salmon gonadotropins (GTH I and GTH II) and the distribution of the receptor(s) were studied through examination of the binding of coho salmon (Oncorhynchus kistuch) GTH I and GTH II to membranes from thecal layers and granulosa cells of salmon ovaries. Purified coho salmon gonadotropins were iodinated by the lactoperoxidase method. Crude membrane preparations were obtained from thecal layers, granulosa cells, and whole ovaries of coho salmon in the postvitellogenic/preovulatory phase. Binding of 125I-GTH I to membranes from thecal layers, granulosa cells, and whole ovaries, and binding of 125I-GTH II to thecal layer cell membranes could be inhibited by both GTHs, but GTH I was more potent than GTH II. In contrast, GTH II was more potent than GTH I in inhibiting 125I-GTH II binding to membranes from granulosa cells and whole ovaries, but the inhibition curves were not parallel. Scatchard plot analysis suggested that there was a single type of receptor in the thecal layers for both GTHs, whereas in the granulosa cells there was more than one type of receptor for both GTHs. Based on these results, a two-receptor model for the postvitellogenic/preovulatory salmon ovary is proposed with the following features: 1) there are two types of gonadotropin receptors in the salmon ovary, type I and type II; 2) the type I receptor binds both GTHs, but with higher affinity for GTH I, whereas the type II receptor is highly specific for GTH II and may have only limited interaction with GTH I; and 3) the type I receptor is present in both thecal cells and granulosa cells, whereas the type II receptor is present in granulosa cells.


Regulation of proto-oncogene expression and deoxyribonucleic acid synthesis in granulosa cells of perifused immature rat ovaries.

The present series of experiments examined the effects of follicle-stimulating hormone (FSH) and insulin (IN) on granulosa cell (GC) proto-oncogene expression and DNA synthesis. In the first study, GCs were harvested from immature rat ovaries after 15, 30, or 60 min of perifusion and DNA synthesis (3H-thymidine incorporation) and proto-oncogene mRNA levels were determined. The presence of c-myc and c-fos proteins was localized within GCs immunocytochemically. GCs of control ovaries exhibited modest levels of DNA synthesis and proto-oncogene expression. FSH/IN not only stimulated DNA synthesis but also increased c-myc, c-fos, and c-jun mRNA levels and the percentage of cells staining for c-fos and c-myc proteins. The protein kinase inhibitor, 2-aminopurine (2-AP), inhibited the FSH/IN-induced increases in c-myc and c-fos mRNA levels, the percentage of cells staining for Myc and Fos protein, and DNA and protein synthesis. The effects of 48 h of perifusion with FSH in the presence or absence of IN were also examined. These treatments were selected because after 48 h of continuous exposure to FSH alone, estradiol-17 beta (E2) secretion is enhanced and 3H-thymidine incorporation is inhibited. Conversely, FSH/IN maintains 3H-thymidine incorporation for up to 48 h of perifusion culture without stimulating E2 (Peluso et al., Endocrinology 1991; 128:191-196). After 48 h of perifusion, both FSH and FSH/IN stimulated c-fos mRNA and protein levels. However, high levels of c-jun mRNA and protein were detected only within GCs of FSH/IN-treated ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of 48-hour infusion of the synthetic oxytocin antagonist, [1-beta-mercapto(beta-(CH2)5)1(OMe)Tyr2,Orn8]-oxytocin, on myometrial activity of pregnant sheep at 139-140 days of gestation.

Currently there is considerable interest in the actions of oxytocin antagonists on the pregnant myometrium. Few studies have been conducted involving long-term infusions of oxytocin antagonists to late-pregnant experimental animals. We set out to determine whether continuous infusion of an oxytocin antagonist ([1-beta-mercapto(beta-(CH2)5)1(OMe)Tyr2,Orn8]-oxytocin) would influence basal levels of myometrial activity of the contracture type and maternal prostaglandins in pregnant sheep. The antagonist was infused into a uterine vein at 80 micrograms.h-1 for 48 h starting at 139 days of gestational age. The antagonist significantly reduced total myometrial electromyogram activity and the frequency of contractures but did not change contracture duration. Antagonist infusion did not produce any significant alterations in maternal carotid or uterine vein 13,14-dihydro-15 keto prostaglandin F2 alpha concentrations. Contractures probably represent an intrinsic instability of the resting membrane potential of uterine muscle and these results suggest that oxytocin may play a role in regulating their frequency in sheep during the last third of gestation.


The perinuclear matrix as a structural element of the mouse sperm nucleus.

The mouse sperm nucleus, after the removal of protamines and DNA, consisted of a skeletal structure that conformed to the original nuclear shape. Sperm were extracted with 1% SDS, and the isolated nuclei, along with the enveloping perinuclear theca, were incubated in 25 mM dithiothreitol, and exposed to different reagents in an effort to displace the protamines, P1 and P2. Protamines, labeled with [3H]arginine, were displaced from the nucleus by CaCl2.MgCl2, but only partially by anionic detergents, monovalent cations, and polyvalent anions. Displacement of P1 and P2 was achieved by digesting the nuclei with DNase I and simultaneously extracting with CaCl2.MgCl2 (3:2; mol:mol) in stepwise increments of 125, 150, 175, 200, and 250 mM. Protamine displacement was concentration-dependent, occurring with an EC50 of approximately 205 mM and with maximal displacement at approximately 250 mM CaCl2.MgCl2. The nucleus was reduced to a skeletal structure consisting of the perinuclear theca and an internal network of transverse fibers. The evidence was consistent with the former being derived from the perforatorium and postacrosomal nuclear sheath (both cytoplasmic structures), whereas the fibers were most likely of nuclear origin. By SDS-PAGE and isoelectric focusing (IEF), perinuclear matrices consisted of greater than or equal to 230 protein spots, with M(r)s in the range of 70,000 to 8000 and pIs of greater than or equal to 7.5 to approximately 4.7, respectively. Monoclonal antibodies prepared against perinuclear matrices bound to specific proteins on IEF immunoblots and, based on light and electron microscopic observations, to discrete domains of the sperm perinuclear theca and nucleus.


Heterogeneity of cell populations that contribute to the formation of primordial follicles in rats.

To study the events that lead to the formation of primordial follicles, pregnant rats were given continuous infusions of [3H]thymidine (3H-TdR) beginning on Days 14-19 of pregnancy (e14-e19) and continuing for 48-120 h. Ovaries from the pups were collected and plastic-embedded histological sections were prepared for autoradiography. The autoradiographs revealed that within the core of the developing ovary were a large number of cells that remained mitotically inactive (failed to incorporate label) from e14 through the day of birth. These unlabeled cells gave rise to the granulosa cells of the first follicles that formed, were located in the medulla of the ovary, and were the first to begin growth. The unlabeled cells did not appear to contribute to the formation of the follicles that formed later in the cortical region of the ovary. When 3H-TdR infusion was begun during late pregnancy, a small subset of the germ cells incorporated label, although the vast majority did not. The labeled germ cells are presumed to represent those that were lagging in their development (had not yet entered meiosis). After ovarian histogenesis was completed during the first week postpartum, the unlabeled ocytes were found concentrated in the core of the ovary, enclosed in the earliest growing follicles; labeled oocytes were found exclusively in the cortex of the ovary, within tiny, quiescent primordial follicles. These observations provide some empirical support for long-held, but heretofore untested, hypotheses concerning early folliculogenesis: that the first follicles that begin to grow are qualitatively different from the remaining follicles in the ovary and that primordial follicles begin to grow in the order in which they were first formed.


Mating induces puberty in the female musk shrew.

Female musk shrews (Suncus murinus), lacking a behavioral estrous cycle, engage in copulatory behavior whenever tested; even during pregnancy, sexual activity can be displayed. Studies suggest that protracted sexual behavior has a functional significance in this species. Virgin females receiving a series of ejaculations over the course of several days are more likely to ovulate and subsequently deliver than animals given the same number of ejaculations over an interval of a few hours. After the virgin mating only one third of the ovaries placed in culture exhibit increased steroidogenesis. In the present set of studies, the hypothesis that the virgin mating induces the onset of puberty, in a manner similar to that in which male-related pheromones induce estrous in rodents, was tested. On the basis of gestational lengths it is evident that females mated three times (on Days 0, 4, and 8) became pregnant in the vast majority of cases as a result of the second and third matings. When the first and second matings were separated by 25 days, 82% of females ovulated in response to the second mating; less than one third of females mated only once ovulated. Finally females were housed across a screen from males, exposed to male urine, or housed alone prior to the virgin mating. In none of these cases did pre-exposure to male-related cues increase ovulation rates in response to the virgin mating. The results show that the virgin mating primes the neuroendocrine system in such a way that the pubertal ovulation can occur in response to subsequent mating.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition of pituitary bioactive prolactin secretion in the male rat by the glucocorticoid agonist decadron phosphate.

In the present studies, the soluble glucocorticoid agonist, decadron phosphate (DEC), was administered i.v. to intact adult male rats in order to evaluate the effects of glucocorticoid receptor stimulation on circulating levels of immunoreactive (ir-) and bioactive (bio-) prolactin (PRL). In light of reports that glucocorticoid-specific receptors exist within the rat brain, additional experiments investigated the effects of intracerebroventricular (i.c.v.) administration of graded doses of the same drug on pituitary ir- and bioPRL secretion. Concentrations of ir- and bioPRL in samples obtained before and after drug treatments were determined by standard PRL radioimmunoassay and the Nb2 rat node lymphoma bioassay, respectively. Rats injected i.v. with 0.5 mg DEC/kg body weight, but not those treated with a tenfold lesser dose, exhibited decreased plasma irPRL concentrations. However, both doses promoted a decline in circulating levels of bioPRL compared to vehicle-treated controls, along with an overall reduction in the plasma bio/irPRL ratio. The magnitude and duration of this drug-induced decline in biopotency of secreted hormone was dose-dependent. While the plasma bio/irPRL ratio was diminished only transiently in rats injected with 0.05 mg DEC/kg, treatment with the higher dose led to a sustained decrease in the plasma bio/irPRL ratio for the duration of the experiment. The current studies also show that intracerebral administration of DEC resulted in dose-dependent alterations in pituitary PRL release. Circulating levels of ir- and bioPRL were not altered in rats injected i.c.v. with 10 ng of DEC, the lowest dose tested.(ABSTRACT TRUNCATED AT 250 WORDS)


Kinetics of follicle growth in the prepubertal gilt.

Follicular growth rates were determined by histological examination of ovaries of five prepubertal gilts following treatment with the stathmokinetic agent colchicine. One ovary from each of five gilts was removed surgically and then colchicine (n = 3) or saline (n = 2) was infused i.v. Precisely 2 h after treatment with colchicine, the remaining ovary was removed. Ovaries were processed for histological analyses and sectioned at 10 microns; every twentieth section was stained with hematoxylin and periodic acid-Schiffe's. Sections were viewed with a projection microscope and individual follicles were measured. Eight classes of follicles were established such that the number of granulosa cells per cross section doubled in each class. Diameters of follicles for each class were as follows: 1) less than 106 microns, 2) 106-148 microns, 3) 148-206 microns, 4) 206-287 microns, 5) 287-400 microns, 6) 400-657 microns, 7) 657-1480 microns, and 8) 1480-3130 microns. A layer of thecal cells was first seen in class 2 follicles, and 76% of class 3 follicles had a thecal layer. Oocyte diameter increased through the first four classes and reached a maximum diameter of approximately 110 microns. Almost all follicles greater than 400 microns had an antrum. Preantral follicles had a lower mitotic index and a higher mitotic time and class time than antral follicles. Growth rate increased with increasing size of follicles. Preantral follicles grew at a rate of 5.2 microns/day whereas antral follicles grew at 313 microns/day.(ABSTRACT TRUNCATED AT 250 WORDS)


Localization of c-fos-like proteins in the mouse endometrium during the peri-implantation period.

Fos, which collectively refers to protein products of the c-fos proto-oncogene family, was immunohistochemically localized in the mouse uterus to ascertain the effects of steroids on Fos expression in the mouse and to determine the course of Fos expression during normal pregnancy and pseudopregnancy. In ovariectomized mice, Fos was induced in luminal and glandular epithelium and an occasional stromal cell following treatment with 100 ng 17 beta-estradiol (E2). Priming with 2 mg progesterone (P4) for 3 days prior to the administration of 100 ng E2 or giving 100 ng E2 and 2 mg P4 concomitantly induced Fos in the luminal and glandular epithelium with enhanced staining in stromal cells. Priming with P4 before giving E2 had the added effect of inducing Fos at an earlier time point. Progesterone alone failed to induce Fos in any cell type in the uteri of ovariectomized animals. On Days 1 and 2 of pregnancy and pseudopregnancy, Fos was detected in the luminal and glandular epithelium and in the stroma. Staining was decreased by Day 3 and was not detectable by the morning of Day 4. No further staining for Fos was found during subsequent days of pseudopregnancy. On the afternoon of Day 4 of pregnancy, Fos was detected in the epithelium and the antimesometrial stroma. On the morning of Day 5, implantation sites and non-site areas were distinguishable, with Fos present in luminal epithelium and antimesometrial stroma in sites, and in just the luminal epithelium in non-sites. Fos remained in the epithelium of non-sites through Day 7 of pregnancy. In sites on the afternoon of Day 5, staining was found in the luminal epithelium and primary decidual zone. On Day 6, staining was found in the secondary decidual zone, but was absent from the primary zone and epithelium. On Day 7, Fos was also detected in the embryonic trophoblast giant cells and the ectoplacental cone. These results demonstrate that E2 and P4 modulate Fos in the mouse endometrium and that Fos is induced during implantation. Significantly, the embryo appears to induce Fos expression on the afternoon of Day 4, while it is still encased in the zona pellucida.


[Functional classification of chronic alcoholic hepatopathy in primary care].

To evaluate the use of Pugh's Classification in assessing patients with diffuse alcoholic Chronic Hepatopathy in the out-patient context. Descriptive-retrospective study. SITE. The Base Health Area of "La Mina" in Barcelona. 143 patients over the age of 14 diagnosed as suffering diffuse chronic Hepatopathy were identified. 37 of these were excluded because of lack of data. The following data were gathered: those relating to semeiology, anamnesis, quantifying alcoholic intake, where the original diagnosis was made, how rapidly the condition evolved, laboratory and complementary explorations' parameters. Out of the 106 patients studied 71 (67%) were classified in group A; 22 (21%) in B and 13 (12%) in C. No significant differences were found in relation to age or sex. Significant differences concerning the speed of the condition's evolution and the average intake in grammes of alcohol per day were observable. The number of hospital admissions increased as the patient's functional stage became worse and complications increased. Pugh's test was shown to be useful in the out-patient context for classifying patients suffering chronic diffuse Hepatopathy in line with the severity of their condition.


[Maternal health knowledge and its use in pediatrics in primary care].

To find the nature of the relationship between the general level of mothers' health knowledge and the over-use of paediatric facilities at the Primary Care level. A crossover study with a control group. SITE. Zaidin-Sur Health Centre (Granada). We selected all the over-users (69) of a Paediatric office and compared them to a random sample, grouped by age, of normal users (82); n = 151. To control confusion, we measured a total of 31 variables, grouped as to need, predisposition and expediting factors. After validating the scales, the average scores for general health understanding and the score for clinical attitudes and knowledge were significantly lower in the over-user group (p = 0.017 and p = 0.000). The scale for attitudes to and knowledge of prevention did not attain any importance (p = 0.104). We worked out a model of multiple logistical regression which included in the equation the scale of clinical attitudes and knowledge (p less than 0.005). We emphasised the importance of mothers' health knowledge to the question of over-use of paediatric facilities. It is desirable to attempt to intervene on this question in order to lessen excessive use of health facilities.


[Minimum sets of data in primary care: an exploration of their feasibility].

To evaluate the different "costings" of several different strategies for gathering a Minimum Set of Data (MSD) at the Primary Care level. A simulation study using a hypothetical MSD with 16 items together with the findings on demand and interventions made by General Physicians in an observational study in Andorra. Different gathering strategies were applied using these data and with the MSD register covering visits as the starting-point. Measurement of the "costing" or work-load involved in the gathering and transmission of each of the items was carried out by means of a nominal group. SITE. General and paediatric consulting-rooms. The simulation was carried out among the catchment population of PAMEM and used data on what was done in 1988. Strategies based on sampling of visits or individuals are those which show a greater reduction in cost (99.3% and 98.1% respectively). Sampling individuals additionally allows for the MSD to be added to during periods of illness. Other options with their respective results are: "gathering information from alternative sources", with a reduction of 22%, "reduction of the number of data to record", with 20% reduction and "separation of stable information" with 18%. The systematic collection of data on each visit has important and much more efficient alternatives. The choice of one of them will depend on how feasible it is to introduce, on the available resources and above all on objectives.


[Prospective study of the development of excess weight in a group of chronic ambulatory patients].

To assess the effect of Health Education on excess weight. Observational crossover study in order to analyse the prevalence of excess weight. Prospective study with intervention. SITE. At the Primary Care level (the Vilanova del Camí Health Centre) in a semi-urban district with 9,173 inhabitants. Patients registered with the chronically ill nursing office in one of three General Medical units. Six patients seen at home and 4 how did not attend personally were not counted. The remaining numbered 87, out of which 17 did not present excess weight (the latter being defined as when there is a ratio of body mass (RBM) superior to 25). For the purpose of the intervention carried out to reduce weight, the 70 obese patients were divided into three groups: no intervention (N), hygienic/dietetic measures (M), and hypocaloric diet (D). The variation of weight and RBM achieved over six months was studied. The parameters for matching data were compared. In group N, a light weight increase of no statistical significance was found. In group M, a highly significant reduction of weight and RBM was observed (p less than 0.001). In group D, weight reduction presented a strong correlation (p less than 0.01) and the reduction in RBM was highly significant (p less than 0.001). An abstentionist attitude to the problem of excess weight cannot be justified, seeing that notable results can be obtained using hygienic/dietetic measures alone.


[Atypical presentations of primary hypothyroidism in primary care].

To present three cases of primary Hypothyroidism with an atypical beginning. Retrospective clinical observations. SITE. Health Centre (Primary Care). Three women (aged 38, 43 and 51) who were studied in the consulting room: one who presented Macrocytosis and Hypercholesterolemia; the second, a bilateral palpebral oedema; and the third, dysfonia unaccompanied by any other symptom of Hypothyroidism. In the three cases, hypercholesterolemia was objectified, I4 diminished and TSH raised; in one of them T3 was normal, while it was low in the remainder. The alteracions observed are frequent in primary Hypothyroidism. But it is rare to find them on their own in the presentation of the clinical picture. Given this pathology's high prevalence, it is important to think of the pathology when presented with these atypical symptoms, and possibly to request the determination of TSH in isolation.


[Chronic pathologies and pharmaceutical expenditure in persons over the age of 65].

For purposes of the present study of people over 65 attending a particular Health Centre, we aimed to discover and inter-relate their morbidity due to chronic illnesses, their frequency of attendance, and the total average outlay of drugs on them. Descriptive crossover study. Information on the clinical histories was gathered over a one-year period (July 1990 to June '91). SITE. Within the Primary Care framework: at the Vite Health Centre in Santiago de Compostela. The population aged 65 or over and with medical records in the said centre. A representative sample of this population (n = 240), obtained by systematic sampling, was worked with. The sample's average age was 73.6 +/- 6.7 years. Arterial Hypertension (AHT) was the most common chronic illness. AHT, LCFA/Asthma, Hyperlipemia and digestive pathology showed significant differences between men and women. The average number of medical consultations per person per year was 11.5 +/- 0.82, with no significant differences as to sex and age. The average outlay per person per year was 27,610 pesetas +/- 38,688, with an average cost per consultation of 2,750 pesetas +/- 5,158.7. The chronic illnesses that produced a higher average total cost were heart diseases. Significant differences were shown between the number of chronic illnesses and the average total cost. The presence, number and nature of chronic illnesses are the factors affecting higher outlay in medication.


A study of cervical cancer in Ethiopian women.

In common with many African countries, Ethiopia has a very limited cytological service, smears are only taken in a hospital or clinic setting and until very recently most had to be sent abroad for analysis. We describe the results of a clinical and cytological investigation of 2111 women attending hospitals and clinics in Addis Ababa: 33 invasive or microinvasive cancers and 10 dysplasias (CIN) were detected. The prevalence of invasive cervical cancer in that population was 15.6/1000. Risk factors were shown to be age over 35 (especially over 50), parity over six (especially more than 10), very low income or subsistence economy, particularly in those from the rural areas, and prostitution. Less significant factors, possibly related to age, were sexual activity of more than 20 years and first coitus before the age of 15; and more than five sexual partners. Most (66%) of women with invasive cancer were at stage 3 or 4.


Measuring proliferation in routine fine needle aspirates. Immunocytochemical detection of bromodeoxyuridine incorporation and Ki-67 expression in breast aspirates.

Two simple quantitative means of measuring tumour proliferation which can be applied to cytological material are described. One method involves immunocytochemical staining of cytological smears prepared from breast aspirates with the monoclonal antibody Ki-67. The other method involves incubation of aspirated material with 5-Bromo-2-deoxyuridine (BrdU). Direct measurement of the S phase of the cell cycle is feasible in breast fine needle aspirates by Bromodeoxyuridine incorporation and subsequent immunocytochemical detection. The proliferation indices obtained correlate with those derived from Ki-67 staining. This technique is suitable for routine use in the assessment of tumour proliferation.


Comparison of four immunochemical methods for the measurement of oestrogen receptor levels in breast cancer.

Four methods of assessing oestrogen receptor (ER) status were compared in 33 patients with operable primary breast cancer. The methods used to assess the ER status were immunocytochemical assay (ER-ICA) of frozen sections, fine needle aspirates and imprint material and enzyme immunoassay (ER-EIA) of tumour tissue. A mean overall ER positivity of 45% (15 out of 33), 41% (13 out of 32) and 21% (six out of 29) was obtained by immunocytochemical (ER-ICA) staining of frozen sections, fine needle aspirates and tumour imprints, respectively, and a mean overall ER positivity of 42% (14 out of 33) was obtained by ER-EIA. The concordance of ER positivity in pairs of data obtained from different method combinations was found to range between 72 and 91%. However, statistically there was no significant difference between the four methods on the basis of the data obtained. Good comparability has been shown between the three tissue analyses and therefore the method of choice is technically not immediately apparent.


Immunocytochemical staining of ovarian tumours with Ov632.

Ov632 monoclonal antibody, said to be highly specific for ovarian tumour tissue, was used to stain ovarian tumour cells in a variety of specimens to determine its sensitivity. Thirteen of the 41 samples investigated were negative suggesting a low sensitivity for Ov632.


Post-irradiation cytology of cervical cancer patients.

The accuracy of cervicovaginal cytology following radiotherapy for cervical cancer is compromised by the anatomical and tissue changes resulting from irradiation. Collection of representative samples may be more difficult, and benign radiation changes, post-irradiation dysplasia, and the frequent occurrence of repair cells and active stromal cells in post-irradiation smears may cause diagnostic problems. Nevertheless, cytology is a valuable tool for the detection of locally recurrent cervical cancer. It is simple and economical to perform at the time of clinical follow-up examination, and may detect occult tumour recurrence. Awareness of the cellular changes resulting from irradiation, and the varied composition of post-irradiation smears may lead to more accurate interpretation of the cytological findings.


'Ciliated' tumour cells in ascitic fluid from two cases of cystadenocarcinoma of the ovary.

We report two cases of primary carcinoma of the ovary in which 'ciliated' adenocarcinoma cells were found in the ascitic fluid. Transmission electron microscopy revealed that these were not true cilia but rather a prolific growth of abnormal microvilli. The cytological findings were compared with the histological appearances of the primary tumour. No ciliated cells were seen in the primary tumour, suggesting that the formation of the microvilli represented an independent proliferation of the cells in the fluid. Special staining reactions for mucin, alkaline phosphatase and epithelial membrane antigen were identical in the primary tumour and the cells in the ascitic fluid.


Metastatic breast cancer presenting as an ovarian cyst: diagnosis by fine needle aspiration cytology.

A fifty-year-old woman with a history of breast cancer presented with a clinically benign ovarian cyst. Laparoscopic aspiration of cyst fluid was performed and the cytology was suspicious of epithelial neoplasia. At subsequent hysterectomy and bilateral salpingo-oophorectomy, the specimen showed extensive metastatic breast carcinoma. Although the cytological examination of fluid from ovarian cysts is often unrewarding, this case shows that metastatic carcinoma may occasionally be diagnosed in this fashion.


A 16 kb small single-copy region separates the plastid DNA inverted repeat of the unicellular red alga Cyanidium caldarium: physical mapping of the IR-flanking regions and nucleotide sequences of the psbD-psbC, rps16, 5S rRNA and rpl21 genes.

The four inverted repeat (IR) flanking regions of the Cyanidium caldarium plastid DNA were cloned. Southern blotting, transcript and sequence analyses of the border regions revealed the psbD-psbC operon and the rps16 gene within the large single-copy region upstream of the 16S rDNA gene and the rpl21 gene downstream of the 5S rDNA within the 16 kb small single-copy region. The size of the IR is ca. 5 kb. The nucleotide sequences of the psbD-psbC, rps16, rpl21 and 5S rRNA genes with the corresponding alignments and physical maps of the regions are presented. Northern analysis revealed a less complex psbD-psbC transcription pattern than has been found in higher plants. Comparisons to other red algal data point to structural diversity within red algal plastid DNA.


Lectin genes from the legume Medicago truncatula.

We report the cloning and characterization of two lectin genes from Medicago truncatula, designated Mtlec1 and Mtlec2. The two genes show a high degree of homology and apparently belong to a small multigene family. Mtlec1 appears to encode a functional lectin with 277 amino acids, whereas Mtlec2 is probably non-functional, since a frameshift mutation (insertion of two nucleotides) leads to premature translation termination after only 98 amino acids. The deduced amino acid sequence of the polypeptide MtLEC1 suggests that this lectin is a metalloprotein with Glc/Man specificity.


Factors affecting the rate of T-DNA transfer from Agrobacterium tumefaciens to Nicotiana glauca plant cells.

Different factors involved in the early steps of the T-DNA transfer process were studied by using a beta-glucuronidase gene (gusA) as a reporter in Nicotiana glauca leaf disc transformation experiments. The levels of transient expression of the gusA gene in leaf discs infected with several strains or vir mutants correlated well with their virulence phenotype, except for virC mutants. The rate of T-DNA transfer was shown to be stimulated in the case of non-oncogenic strains by the co-transfer of small amounts of oncogenic genes. It was found that the location of the T-DNA in the Agrobacterium genome affected the T-DNA transfer rate especially in virC mutants. The virC mutants transferred the gusA-containing T-DNA located on a binary vector more efficiently than the oncogenic T-DNA of the Ti plasmid. Although wild-type strains induced high levels of gusA expression early after infection, the gusA expression appeared to be lost late after infection in the infected leaf discs. In contrast, in leaf discs infected by virC mutants the level of gusA expression increased steadily in time. A model explaining these results is presented.


Molecular analysis of a cruciferin storage protein gene family of Brassica napus.

We have isolated a five-member gene subfamily which encodes cruciferin, a legumin-like 12S storage protein of Brassica napus L., and have analyzed the structure and expression of the family members in developing embryos. Sequence analysis has shown that the coding regions of all five genes are highly similar, with the two most divergent members of the family retaining 89% sequence identity. The analysis of this cruciferin gene family's expression indicates that the developmental pattern of expression of each gene is similar, and the steady-state mRNA levels of each gene are approximately equivalent to each other at all developmental stages.


Information contents and dinucleotide compositions of plant intron sequences vary with evolutionary origin.

The DNA sequence composition of 526 dicot and 345 monocot intron sequences have been characterized using computational methods. Splice site information content and bulk intron and exon dinucleotide composition were determined. Positions 4 and 5 of 5' splice sites contain different statistically significant levels of information in the two groups. Basal levels of information in introns are higher in dicots than in monocots. Two dinucleotide groups, WW (AA, AU, UA, UU) and SS (CC, CG, GC, GG) have significantly different frequencies in exons and introns of the two plant groups. These results suggest that the mechanisms of splice-site recognition and binding may differ between dicot and monocot plants.


Direct sequencing of tobacco chloroplast genome by the polymerase chain reaction.

We have developed a polymerase chain reaction (PCR) method for sequencing of tobacco chloroplast genome. In a mixture containing chloroplast DNA, 5'-end-labeled oligonucleotide primer, Taq DNA polymerase and reaction buffer, we were able to sequence a segment of chloroplast 16S rRNA gene. The results showed that the 750 bp of DNA sequenced were identical to the sequence reported, indicating that direct sequencing method that we have developed is useful for the sequencing of chloroplast genome. To analyze the chloroplast genome more rapidly in those in vitro grown plantlets, we also developed a simple method which is applicable for the amplifications and sequencing of chloroplast 16S rRNA fragment from either 0.15 g of tobacco leaf or stem tissue. The readable sequences obtained from the presented methods were consistent with the published sequence.


Sequence of an oleosin cDNA from Brassica napus.

Antibodies raised against purified rapeseed 19 kDa oleosin protein were used to screen an embryo-derived lambda gt11 expression library from Brassica napus. A near full-length cDNA clone, BnV, was isolated. The 781 bp cDNA contained an open reading frame of 549 bp followed by an untranslated region of 222 bp and a poly(A) region of 10 bp. Comparisons between this cDNA and a different oleosin cDNA previously isolated from the same library showed high degrees of sequence similarity in the central domain region and in the 3' untranslated region. Sequence similarities between the derived protein sequence of this cDNA and all other known oleosin protein sequences are discussed.


Aleurone nuclear proteins bind to similar elements in the promoter regions of two gibberellin-regulated alpha-amylase genes.

Binding of nuclear proteins from wild oat aleurone protoplasts to the promoter regions of two gibberellin-regulated wheat alpha-amylase genes (alpha-Amy1/18 and alpha-Amy2/54) has been studied by gel retardation and DNase 1 footprinting. Gel retardation studies using 300-430 bp fragments of the promoters showed similar binding characteristics with nuclear extracts from both gibberellin A1-treated and untreated protoplasts. DNase 1 footprints localised binding of nuclear proteins from gibberellin A1-treated aleurone protoplasts to regions in both promoters. Similar sequence elements in the promoter regions of both genes were protected from digestion although the location and number of footprints in each promoter region were different. Each footprint contained either a sequence similar to the cAMP and/or phorbol ester response elements, or a hyphenated palindrome sequence. The presence of cAMP and/or phorbol ester response element-like sequences in the footprints suggests that transcription factors of the bZIP type may be involved in the expression of alpha-amylase genes in aleurone cells. Footprints containing hyphenated palindrome sequences, found in the promoter regions of both genes, suggest the possible involvement of other classes of transcription factor. The conserved alpha-amylase promoter sequence TAA-CAGA was also shown to bind nuclear protein in the alpha-Amy2/54 promoter. These observations are discussed in relation to alpha-amylase gene expression in aleurone and to functional data concerning these genes.


Localisation of cis elements in the promoter of a wheat alpha-Amy2 gene.

A functional analysis of the promoter from the wheat alpha-amylase gene alpha-Amy2/54 is described. Mutant alpha-Amy2/54 promoters containing replacements or deletions were constructed and their ability to direct expression of the reporter gene beta-glucuronidase (GUS) in gibberellin-responsive oat aleurone protoplasts analysed. Chimaeric promoters using regions of the cauliflower mosaic virus (CaMV) 35S and alpha-Amy2/54 promoters were also analysed. The results suggest that at least three regions within the alpha-Amy2/54 promoter contain cis elements that are necessary for high-level gibberellin-regulated transcription. Fusion of 1.8 kb of promoter sequence upstream from -117 bp to a minimal (-55 CaMV 35S) promoter gave rise to hormone-independent expression implying that the region 3' to -117 bp contains an element which represses transcription in the absence of gibberellin or presence of abscisic acid.


Sequence and transcription analysis of mitochondrial plasmids isolated from cytoplasmic male-sterile lines of Vicia faba.

Three mitochondrial plasmids (1704, 1695 and 1478 bp) were isolated from sterile cytoplasms of Vicia faba L. and cloned into a bacterial plasmid vector. Their nucleotide sequence was found to be 99 to 100% homologous to their counterparts isolated from a fertile cytoplasm (J.A. Wahleithner and D.R. Wolstenholme, Curr Genet 12 (1987) 55-76). Several overlapping transcripts were localized in the region which is unique to each of the three plasmids. S1 nuclease mapping indicated for all of them several 3' termini but a unique 5' boundary which was located downstream of the consensus sequence CNTAAGTGANNNNNGAA also found at the transcript 5' boundary of other plant mitochondrial plasmids. Southern blot hybridization with nuclear DNA indicated the presence of nuclear sequences homologous to each plasmid.


Genetic transformation of Norway spruce (Picea abies (L.) Karst) using somatic embryo explants by microprojectile bombardment.

Stable transformation of Norway spruce tissue has been obtained following bombardment of mature somatic embryos with pRT99gus, a plasmid that contains neo coding for NPTII, and gusA, coding for beta-glucuronidase, both fused to the CaMV 35S promoter. At least 8 lines have been stably transformed (over 15 months in culture) following bombardment and selection on kanamycin. Polymerase chain reaction analyses showed a high frequency of cotransformation of the gusA and neo genes. The frequency of coexpression of the selected and unselected markers was 100%. DNA/DNA hybridization of one transformed line provided conclusive evidence of stable integration and showed copy numbers of over 10 plasmid sequences per genome. None of the transformed lines has remained embryogenic.


The legumin boxes and the 3' part of a soybean beta-conglycinin promoter are involved in seed gene expression in transgenic tobacco plants.

beta-conglycinin is one of the major seed storage proteins in soybean. It is composed of three subunits, namely alpha, alpha' and beta. The expression of beta-conglycinin is highly regulated, being restricted to the embryo during the mid-maturation phase of embryogeny. Two series of constructs were made with the alpha' subunit promoter and the GUS reporter gene to investigate the cis-acting elements involved in the regulated expression of this promoter. The activity of each construct was tested in transgenic tobacco plants. In the first series of constructs, we checked if the 'legumin box', a sequence found in most legume seed storage protein genes as well as in other seed-specific genes, is involved in the regulated expression of the alpha' subunit of the beta-conglycinin gene in tobacco. To this end, both copies of the alpha' subunit promoter legumin boxes were mutagenized in vitro. The transcriptional activity of the single mutants and the double mutant were compared with that of the wild-type promoter. Our results show that the legumin boxes act together to increase transcription of the beta-conglycinin alpha' subunit gene by about a factor of ten. This is the first demonstration of a function for the legumin box in transcriptional regulation. In the second series of experiments, we wished to determine if the 3' part of the promoter (the CCAAT and TATAA region) contains important regulatory elements. We found that this small fragment (-82 to +13 bp) can confer by itself a low level of seed-specific gene expression. Chimaeric promoters constructed from parts of the alpha' subunit promoter and of the constitutive CaMV 35S promoter were also analysed. These constructs also revealed the importance of the CCAAT and TATAA region of the alpha' subunit promoter in seed-specific gene expression.


Constitutive expression of the beta-phaseolin gene in different tissues of transgenic alfalfa does not ensure phaseolin accumulation in non-seed tissue.

Phaseolin is a glycoprotein that constitutes the major storage protein in bean seeds. The phaseolin gene promoters function in a seed-specific manner. In an attempt to understand if events following transcription of the gene also contribute to the seed-specific accumulation of the phaseolin protein, we studied the effect of substituting the constitutive CaMV-35S promoter for the beta-phaseolin gene promoter on expression of the phaseolin gene in different plant organs. A chimeric gene consisting of the 35S promoter, the coding sequence of the beta-phaseolin gene (all five introns and six exons) and the 3'-flanking region of the beta-phaseolin gene, was introduced into alfalfa via Agrobacterium tumefaciens. While all organs examined shared high levels of phaseolin transcripts, the only organ that showed significant accumulation of the phaseolin protein were the mature seeds. Co-migration of the major immunoreactive polypeptides from the non-seed organs with the authentic beta-phaseolin polypeptides on SDS-PAGE indicates that the protein in non-seed organs undergoes correct post-translational processing and modification, but are more unstable in a non-seed environment.


Characterization and expression of U1snRNA genes from potato.

U1 small nuclear RNAs (U1snRNAs) occur in the nucleus of plants and animals where, complexed with several proteins in the form of U1 small nuclear ribonucleoprotein particles (U1snRNPs), they play an important role in precursor messenger RNA (pre-mRNA) splicing. Ten potato U1snRNA genes have been isolated on two genomic clones illustrating the clustering of this multigene family on the potato genome. Based on both the sequence of their coding regions and upstream regulatory elements, seven of the genes are potentially functional. The other three genes were pseudogenes with defective promoter or coding region sequences. Analysis of expression of individual cloned U1snRNA genes in transfected tobacco protoplasts was impossible due to the similarity of U1snRNA sequences in tobacco. However, by marking the coding regions with oligonucleotides or constructing chimaeric genes consisting of a potato U1snRNA promoter region and maize U5snRNA coding region, three of the U1 promoter regions were shown to be transcriptionally active.


Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNA polymerase III.

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3'-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to alpha-amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.


A model for quality assessment in cervical cytology used as a screening test.

Although many reliability studies on cervical cytology have been carried out, measurements of sensitivity and specificity have rarely been made since biopsies are not often performed on patients with a negative smear result. This screening assessment was performed over 3 years, using a database of 230,167 smears from 177,051 women. It would seem that cervical cytology has a high specificity (over 99%) but a relatively low sensitivity (61%). Values for sensitivity (exceeding 95%) and specificity (exceeding 99%) for invasive carcinoma should be regarded as reasonably accurate as all incident cases of symptomatic cervical carcinoma are recorded in the Burgundy register. The sensitivity (57%) for pre-invasive lesions is underestimated while their prevalence is overestimated: the lack of organized screening leads to the loss of prevalent cases. The predictive value of a positive smear is 76% for moderate-severe dysplasia, 85% for in situ carcinoma and over 95% for invasive carcinoma.


AT-rich promoter elements of soybean heat shock gene Gmhsp17.5E bind two distinct sets of nuclear proteins in vitro.

A 33 bp double-stranded oligonucleotide homologous to two AT-rich sequences located upstream (-907 to -889 and -843 to -826) to the start of transcription of heat shock gene Gmhsp17.5E of soybean stimulated transcription when placed 5' to a truncated (-140) maize Adh1 promoter. The chimeric promoter was assayed in vivo utilizing anaerobically stressed sunflower tumors transformed by a pTi-based vector of Agrobacterium tumefaciens. Nuclear proteins extracted from soybean plumules were shown to bind double-stranded oligonucleotides homologous to AT-rich sequences in the 5' flanking regions of soybean beta-conglycinin, lectin, leghemoglobin and heat shock genes. These proteins were also shown to bind AT-rich probes homologous to homeobox protein binding sites from the Antennapedia and engrailed/fushi tarazu genes of Drosophila. Binding activity specific for AT-rich sequences showed a wide distribution among various plant organs and species. Preliminary characterization indicated that two sets of nuclear proteins from soybean bind AT-rich DNA sequences: a diverse high-molecular-weight (ca. 46-69 kDa) group, and a low-molecular-weight (23 and 32 kDa) group of proteins. The latter meets the operational criteria for high-mobility group proteins (HMGs).


The Flemish Centre for the Study of Perinatal Epidemiology and its registry.

The Flemish Centre for the Study of Perinatal Epidemiology was formally established in 1986. Its objectives are the promotion of perinatal epidemiology and the study of maternal and perinatal mortality and morbidity. One of the means to accomplish these objectives was the creation of a databank of perinatal medicine. The registry at present covers almost 80% of all deliveries in Flanders. The registry indicates a maternal death rate of 5.8/100,000 living births whereas the Belgian official national statistics indicate a maternal death rate of 2.8/100,000. This means that either the matter is under reported at the national level or there is a real problem in the Flemish part of the country. The perinatal death rate varies among the participating services form 3.9 to 22.4%.


What makes outpatient attendance worthwhile for patients?

All patients attending 10 general medical and general surgical outpatient clinics at a provincial teaching hospital were surveyed during a 1-week period. Questionnaires administered and completed by the patients at the time of the visit were used to establish the factors most closely associated with patients feeling that their consultations had been "very worthwhile". These were: consultations lasting more than 10 min, having received advice from a specialist and having been reassured. The study may provide a basis for a process of medical audit in outpatient departments and, thus, more appropriate services.


Need for and influence of feedback from the Finnish birth register to data providers.

Data for the Finnish medical birth register (established 1987) are collected by local hospital personnel as a part of their routine work. The purpose of this study was to study the need of personnel for feedback and the impact of feedback on later data quality. Furthermore, we studied whether such feedback tends to modify extreme cesarean section rates. Data on attitudes towards the birth register and on the need for feedback of data providers were collected through interviews and observations. In March 1988, an information package describing births, birth procedures and infant outcomes in each hospital compared with other hospitals was sent to a random stratified sample of 26 hospitals out of a total of 53. Opinions of the package were obtained by questionnaire from 104 physicians and nurses (82% response rate). Most hospital personnel, especially physicians, had negative attitudes towards the birth register. Comparison of the hospitals which had received feedback with other hospitals in terms of quality of data furnished in 1987 and 1988 suggested that feedback may improve the technical quality of data. There was no evidence, however, that feedback caused hospitals to change their practices in regard to cesarean sections.


Quality assurance in urine analysis.

An Australian quality control (QC) system was tested on its ability to detect analytical errors in calcium, creatinine, oxalate, phosphate and uric acid determinations in urine. This QC system revealed that our methods for calcium and urate worked well. The oxalate values were systematically higher than the assigned values and the random error was too high. Phosphate and creatinine had a marked positive deviation in the higher range of concentration but agreed better in the medium and lower ranges. The findings emphasize the need for external QC systems in urine analysis. We also discuss specific problems with urine QC compared with conventional QC for serum, the theoretical basis of creatinine-corrected urinary analytes and criteria for rejection and warning limits. We conclude that QC of urine analysis is as important as that of serum and that such programs should be used in concordance with serum QC programs.


Group judgments of appropriateness: the effect of panel composition.

The current interest in the development of practice guidelines raises an important question about the effect of expert panel composition on the outcome of the guideline development process. We compared the ratings of appropriateness of indications for carotid endarterectomy produced by two panels: an all-surgical panel and a "balanced" panel composed of four surgeons, two neurologists, and one specialist each from family practice, internal medicine, and radiology. We then compared the effect of the two sets of ratings when used to evaluate 1302 patients who had undergone carotid endarterectomy. The all-surgical panel found more indications "appropriate" (24 versus 14%) and fewer indications "inappropriate" (61 versus 70%) than the balanced panel (p less than 0.01). The all-surgical panel also more often reached agreement. When ratings were applied to patients, 70% were appropriate by the all-surgical criteria versus 38% by the balanced panel, while 19% of the operations were inappropriate by all-surgical criteria, versus 31% by the balanced panel ratings. However, the percentage of procedures judged "inappropriate with agreement" was 15% for all-surgical and 16% for the balanced panel. We conclude that the all-surgical panel was more likely to favor operative treatment for carotid disease than the multispecialty panel, but that concensus judgments of inappropriateness by the two panels were very similar.


Reactions of doctors to various forms of feedback designed to improve the sampling quality of cervical smears.

Three different forms of feedback designed to improve the sampling quality of cervical smears were tested in a randomized controlled trial among 179 physicians. The reactions of the doctors to the various interventions are reported as they appeared from their answers in a questionnaire. The influence of the interventions on the employed sampling technique is described on the basis of their answers to a telephone inquiry. The inclusion in laboratory protocols of an evaluation of the sampling quality of the submitted smears was largely appreciated, as was specific advice offered on indication. Monthly overviews comparing the performance of the individual doctor to that of his peers were esteemed by three out of four. Further information, help or training was requested more frequently than average by physicians obtaining inadequately sampled smears relatively often. The number of doctors using the combined spatula and Cytobrush method as sampling technique doubled during the study period.


Medical audit and quality of care--a new English initiative.

In 1989 the Government in the UK announced a fundamental review of the financing of the National Health Service (NHS). Before the review, health authorities were responsible for both establishing need and providing services. Health Authorities are now allocated resources with which they commission services from managerially independent provider units. In the discussion that preceded the review concern was expressed about standards of clinical care given by doctors. It was recognised that these were professional issues which could not be directly addressed through the commissioning process, therefore within its NHS review proposals, the Government made clear its expectation that all doctors working in the NHS should participate in medical audit. This was defined as a systematic peer review of their work, including procedures used for diagnosis and treatment, the use of resources and the resulting outcome for patients. Managers should be provided with regular general reports of audit activity. These proposals for medical audit have been well received by the medical profession, who are now working closely with the Government to put them into practice.


The quality improvement system in the hospitals of Padua (Italy).

A quality improvement system has been established in 1989 in the hospital network of Padua and its organization is described. Three selected experiences are reported. (1) Appropriateness of the use of human albumin. After the assessment of the clinical policy, new guidelines were experimentally introduced and an evaluation after 3 months has shown a decrease of the total number of prescriptions (25%) and of inappropriate indications (9% vs 40.1%). (2) Urinary Tract Infections (UTI) and indwelling catheterization. The study showed 49% of conditions related to UTI and some corrigible inadequacies in the process of care: 37.2% of indications were probably not justified; 40% of patients who did not undergo urineculture had indications and 13% who underwent urineculture had no indications to the test. Guidelines for appropriate indications and a continuing education programme have been introduced. (3) Falls by hospitalized patients. The patient fall rate was 0.3/1000. As the reporting system showed inaccuracies (for example, the severity of injury was not collected in 34% of cases), a new notification form was introduced in 1991.


The retinoblastoma protein and the cell cycle.

Although mutations of the retinoblastoma gene (RB1) contribute to malignant progression in many types of tumor, the role of RB1 mutation in cancer initiation is highly restricted to the rare embryonic tumor, retinoblastoma. However, RB1 is expressed and its product, p110RB1, is regulated through the cell cycle in many tissues. This apparent paradox may be clarified by the emerging evidence that p110RB1 functions as a regulator of transcription of cell cycle genes. Tissue specific effects may be determined by the cellular proteins that interact with p110RB1, or by cell type-specific expression of target genes regulated by p110RB1.


Neurofibromatosis: from gene to phenotype.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disease which predominantly affects tissues derived from neural ectoderm. The gene has recently been cloned, and the biochemistry and cell biology of normal and mutant forms is gradually being elucidated. Despite the success of cloning the gene, there is a long way to go before the different features of the disease phenotype can be explained. Some of the questions which arise in the attempt to link the mutation to the phenotype are considered, with possible ways to approach them.


The role of tumour suppressor genes in familial cancer.

Many forms of cancer have a higher incidence in relatives of patients than in the general population, and some show Mendelian inheritance. Although individuals genetically predisposed to cancer represent a minority of all cancer patients, the genetic basis for their disease has profound significance. These familial cases provide strong evidence that germline alterations can contribute to cancer. They also provide an ideal opportunity to identify and isolate the genes mutated in common cancers. Products of the tumour suppressor genes have been implicated in several hereditary forms of cancer. The distinct functions of these proteins and their roles in familial cancer will be discussed.


Familial breast cancer.

Familial clustering of breast cancer has been recognised for over a century but until recently a genetic basis has been suspected rather than proven. Epidemiological studies have tended to support the view that an autosomal dominant gene, with high but incomplete penetrance, accounts for most breast cancer families. However, it is likely that several different predisposing genes are present within most populations. Difficulties arise in a conventional 'linkage mapping' approach to identifying these genes, first, because it is not clear that genetically homogeneous groups of families can be recognised on the basis, for example, of mean age of onset or pattern of other cancers within the kindred and, second, because breast cancer is so common (affecting almost one in twelve women) that large affected kindreds are likely to include an admixture of sporadic (non-genetic) cases. Cytogenetic and 'Loss of Heterozygosity' (LOH) studies in sporadic breast cancers have pointed to several candidate loci for breast cancer genes but there is no clear consensus from these two approaches that might direct attention to any prime target region. Recent reports of tight linkage between familial breast cancer (early onset) and breast/ovarian cancer (regardless of mean age of onset) and a locus on chromosome 17q21 defined by the anonymous probe CMM86, have not been confirmed in detail but have led to the identification of a locus some 15 Mb centromeric of CMM86 that gives a high positive lod at very low recombination fraction in fifteen Edinburgh breast and breast/ovarian cancer families. The disease in the majority of such families therefore appears to be attributable to a mutant gene at 17q12-21. A much smaller proportion of familial breast cancer is accounted for by mutations in the p53 gene (17p13). Not all such families fulfil the criteria for Li-Fraumeni syndrome and not all of the inherited mutations lie within exon 7 of p53. Counselling of members of breast cancer families becomes more exacting as these genetic lesions are identified. It is essential to extend the collection of data and tissue (blood or fixed pathology material) as widely as possible to confirm linkage to a specific locus within each individual kindred, to define the precise mutation and to establish the cancer phenotype and its penetrance. In the course of these studies a substantial population of women at high risk of breast (and other) cancer will be identified. Resources should be directed to this population so that optimum procedures for screening and prevention can be developed.


Genome imprinting and cancer genetics.

Parent-of-origin-dependent modification of the genome (genome imprinting) is thought to be involved in the formation of several types of human cancer. Simple modifications of Knudson's two-hit model give rise to two types of predictions with respect to the genetic behavior of such diseases. In the first, rare alleles of modifier genes will give rise to the retention of the same parent's tumor suppressor alleles in sporadic cases. In the second, other variants of the modifier genes will result in failure to establish linkage between tumor suppressor genes and disease predisposition in familial cases. Both of these predictions are fulfilled by experiment. Additional modifications of existing models are predicted to result in more complex patterns of inheritance. The demonstration of such patterns in human pedigrees will require an extremely sophisticated level of genetic analysis.


Genetic resolution of susceptibility to cancer--new perspectives.

Neoplastic development is under strong genetic control, which results in large differences in tumor susceptibility between individuals of the same species. These differences are caused by tumor susceptibility genes (TSGs). The characteristic features of TSGs are frequent polymorphism, influence on specific aspects of the tumorigenic process, and tissue specificity. In contrast to cellular oncogenes and tumor suppressor genes, which in most instances require somatic alterations to become engaged in the tumorigenic process, the allele-specific effects of TSGs are encoded in the germ-line. Until recently, the TSGs have remained virtually unknown despite many efforts to map them. Their multiplicity and incomplete penetrance defied the available genetic methods. Therefore a novel genetic tool, the Recombinant Congenic Strain (RCS) has been developed in the mouse (P. Demant and A.A.M Hart, Immunogenetics 24:416-422), which allows dissection of multigenic control and mapping of the genes contributing to it. When applied to colon tumors, the RCS revealed separate genetic control for tumor numbers and tumor size, and allowed mapping of several TSGs. The presently available RC strains allow the genetic and functional dissection of virtually all major types of mouse tumors. This opens the possibility to map, within a relatively short period, the TSGs for various organs, to study the functional effects of individual TSGs and the possible interactions of this new class of genes with the known oncogenes and tumor suppressor genes. The extensive information about the chromosomal homologies between man and mouse will facilitate the search for their human counterparts once the TSGs in mouse are mapped.


Stem cell regulation, tissue ontogeny, and oncogenic events.

The number of necessary oncogenic events is a function of tissue ontogeny. A minimum of two events appears to suffice for certain embryonal tumors, leukemias, and lymphomas for which the target tissues normally show stem cell proliferation. Other tumors, including those featured in the Li-Fraumeni syndrome, arise in target tissues whose stem cells are conditionally stimulated to proliferate, as in response to hormones, and involve more events. The most complex cancers include most carcinomas, which arise in renewal tissues whose stem cells do not normally proliferate. The number of necessary oncogenic events appears to increase with the number of controls on proliferation.


The effect of phlebotomy on serum erythropoietin levels in normal healthy subjects.

We evaluated endogenous serum erythropoietin (Epo) levels in 14 normal subjects (eight males and six females) after a single 400-ml phlebotomy. The subjects were followed up for 56 days. The hemoglobin (Hb) values of both males and females decreased to a nadir on days 3 to 7 post-phlebotomy. Hb values gradually increased, but did not completely recover to pre-phlebotomy levels by day 56. Serum Epo levels increased after 6 h post-phlebotomy, to 20.1 +/- 5.4 (mU/ml) in males and 20.7 +/- 7.0 in females, from the pre-phlebotomy levels of 14.6 +/- 4.0 in males and 13.4 +/- 4.1 in females, respectively. Epo levels continued to increase to peak levels of 25.5 +/- 6.3 in males and 28.7 +/- 11.5 in females on days 7 to 14 and thereafter decreased until day 56. Thus, the Epo response to a 400-ml phlebotomy was relatively small in magnitude and was not sufficient to initiate a significant increase in erythropoiesis. This finding suggests that the administration of recombinant human erythropoietin (rHu-Epo) may be effective for the prompt correction of anemia induced by autologous blood donation and for increasing the volume of predonated autologous blood.


Pernicious anemia in Turkey.

Pernicious anemia patients who were diagnosed during a 5-year period in Cukurova University Hospital, Adana, Turkey were reviewed. Of approximately 200 new patients per year accepted by the Hematology Unit 44 were diagnosed as having pernicious anemia. There were 30 males and 14 females. The mean age for men was 49.14 +/- 18.11 and that for women was 40.00 +/- 14.05. Both values and the mean age overall were lower than the reported mean age for Whites, Blacks and Latin Americans living in the United States.


Ionizing radiation increases TNF/cachectin production by human peripheral blood mononuclear cells in vitro.

Human peripheral blood mononuclear cells (PBMC) and monocytes produce a large amount of tumor necrosis factor/cachectin (TNF) in vitro. Our data suggest that X-irradiation can increase TNF production by these cells. After 900 cGy irradiation there were 7-fold (24 h incubation) and 2.3-fold (48 h incubation) increases of TNF production by stimulated PBMC. The irradiation also increased TNF production by both stimulated and unstimulated human monocytes. These findings suggest that TNF overproduction can play an important role in the pathogenesis of radiation disease.


Identification and characterization of specific receptors for the LD78 cytokine.

LD78 is a small secreted protein that has a sequence similar to a number of other polypeptides, including murine macrophage inflammatory protein 1 alpha (MIP-1 alpha), interleukin 8 (IL-8), Act-2, monocyte chemoattractant protein 1 (MCP-1), and others. These polypeptides are members of a novel cytokine superfamily that is involved in the inflammatory response, wound healing, hematopoiesis, and tumorigenesis. Specific receptors for purified clonal LD78 protein were measured using four cell lines (HL-60, U937, Jurkat, and MJ). 125I-labeled recombinant LD78 bound most efficiently to U937 cells. We therefore characterized the receptors as being on the surface of U937 cells. Binding reached an equilibrium after incubation for 60 min at 4 degrees C. Scatchard analysis showed that there were two classes of binding sites on U937 cells, high affinity sites (Kd = 5.3 x 10(-9) M) and low affinity sites (Kd = 9.3 x 10(-8) M), with the average number of binding sites per cell being approximately 30,000 and approximately 90,000, respectively. These receptors for LD78 were distinct from the receptors for gamma-IFN and for IL-8. SDS-PAGE analysis of chemically crosslinked 125I-labeled LD78 receptor complexes identified a single band of 52 kDa. The ability to detect specific LD78 receptors should prove valuable in efforts to molecularly clone these receptors and to dissect the biological actions of LD78.


Serial changes of the serum macrophage colony-stimulating factor level after cytoreductive chemotherapy.

To investigate the physiologic role of macrophage colony-stimulating factor (M-CSF) in hematological recovery from bone marrow hypoplasia, we used an enzyme-linked immunosorbent assay to measure serial changes of the serum M-CSF level during 25 intensification chemotherapy courses given to seven patients with acute non-lymphocytic leukemia who were in complete remission. Three M-CSF peaks were observed during therapy: the first peak was during or just after chemotherapy, the second peak was around the leukocyte nadir, and the third peak coincided with a rapid increase in the monocyte count. We could find no significant correlation between the height of the second peak and the time from the initiation of therapy to hematological recovery. On the other hand, there was a significant positive correlation between the height of the second peak and the interval from the last day of chemotherapy to the peak (r = 0.62, p = 0.001), and there was a significant negative correlation between the peak height and the time from the peak until hematological recovery (defined as a neutrophil count of over 500/microliters (r = -0.63, p = 0.001) and a leukocyte count of over 1,000/microliters (r = 0.55, p = 0.008)). However, we found only a weak correlation between the peak height and monocyte recovery. These data suggest that increased M-CSF levels lead to the stimulation of granulocyte progenitors, and that we can predict the time of neutrophil recovery by monitoring the serum M-CSF level and finding its peak.


Treatment of chronic neutropenia of childhood responsive to cyclosporin A in vitro and in vivo.

The clinical course of a 17-year-old patient who suffered from chronic neutropenia without cyclic variation since the age of 2 is presented. The bone marrow showed absent granulopoiesis and yielded very few colony-forming units (CFU-GM) in vitro with maturation up to segmented neutrophils. Incubation with cyclosporin A (CyA) increased CFU-GM markedly. Such an increase was not found after incubation of normal bone marrow with CyA in vitro. The patient also responded to CyA in vivo and maintained adequate granulocyte counts for 7 months when she became neutropenic again. She subsequently responded to high doses of prednisolone. The clinical course and bone marrow studies suggest that the defective granulopoiesis is due to an immunologically mediated mechanism sensitive to CyA and prednisolone. Other findings in this patient, such as impaired natural killer (NK) cell activity and random and chemotactic leukocyte motility, could further point to an imbalance in the regulation of hematopoiesis. Neutropenia, NK cell defect and impaired chemotaxis may be pathogenetically connected.


Bone marrow eosinophilia as a prognostic indicator in acute myelogenous leukemia with 8;21 translocation.

The pretreatment characteristics of nine acute myelogenous leukemia (AML) patients with 8;21 translocation were evaluated to assess their value as prognostic indicators. All patients had AML-M2 according to French-American-British classification. The percentage of eosinophils in the bone marrow (EO/BM) correlated negatively with the percentage of blast cells in the peripheral blood (BL/PB) (p less than 0.05) and positively with complete remission duration (p less than 0.01). Three of the six patients with EO/BM under 5% showed aggressive clinical courses complicated with extra marrow lesions, while the patient with the highest EO/BM was the only one who survived for 5 years. These findings suggest that bone marrow eosinophilia could be useful as a prognostic indicator in AML patients with t(8;21).


Possible mechanisms for the elevation of serum beta 2-microglobulin levels in adult T-cell leukemia.

In our previous study, we found that serum beta 2-microglobulin (beta 2M) levels were elevated in the active, but not in the inactive, phase of adult T-cell leukemia (ATL), suggesting a correlation between the beta 2M level and the clinical severity of this disease. In this study we examined the mechanisms underlying the elevation of serum beta 2M levels in ATL. First, the production of beta 2M by ATL cells was investigated in vitro. High levels of beta 2M were detected in the conditioned culture medium (CM) of ATL cells from seven out of nine patients. Second, we assessed the effects of the CM on the release of beta 2M by three human cell lines unrelated to ATL (NCTC 2544, Chang liver, and L 132; originating from the skin, the liver, and the fetal lung, respectively). Most of the CM definitely promoted beta 2M production by these cell lines. beta 2M production by the cell lines was markedly promoted by exogenous interferon-gamma (IFN-gamma), a well-known potent inducer of class I HLA antigen expression. We then investigated whether an antibody directed against IFN-gamma could attenuate the activity of three ATL CM. The anti-IFN-gamma antibody reduced the stimulatory activity of the CM to 28-65% of the original level, but did not affect basal beta 2M production by these cell lines. These data suggest that there are at least two mechanisms causing the elevation of serum beta 2M levels in ATL; direct production by tumor cells, and production by non-malignant cells that is mediated via humoral factors secreted by the ATL cells.(ABSTRACT TRUNCATED AT 250 WORDS)