Current concepts in embryonic craniofacial development.

Embryology mirrors phylogeny. The phenotypic expression of the genome is the result of differential gene transcription, the critically timed turning on and off of specific genes by transcription factors to produce cyto-, histo-, and morpho-differentiation that fleetingly reflects evolutionary stages of development during ontogeny. Hox genes regulate transcription of other structural genes and are responsible for patterning of the facial primordia. Cephalic development involves extremely complex morphogenetic mechanisms built on conserved elements that have undergone enormous evolutionary changes. Transient expression of phylogenetic origins characterize ontogeny and are reflected in defective development that may be due to inappropriate expression of Hox genes or distorted or disrupted epignetic processes. The mechanisms by which genetic information is transformed into morphological patterning by the actions of growth factors, morphogenes, and receptors are currently being identified. Biochemical, immunological, and allometric analyses of embryos and fetuses in experimental and descriptive studies are elucidating details of units of craniofacial morphogenesis--faciogenesis, palatogenesis, gnathogenesis, odontogenesis. Three-dimensional model computer-assisted reconstruction of sectioned embryos and fetuses provides a further technique for understanding the complex configurations of tissue migratory patterns and growth sites that account for normal and abnormal craniofaciogenesis.


A model of the adrenergic beta-2 receptor and binding sites for agonist and antagonist.

From the known experimental data on adrenergic ligands and beta-2 receptor a model for the ligand binding sites was proposed and built. Agonist and antagonist have overlapping but different binding sites. The model correlates well with the structural information known from a series of adrenergic ligands.


Synthesis and cardioselective beta-adrenergic antagonist activity of quinolyloxypropanolamines.

The synthesis and beta-adrenergic antagonist activities of (2R,2S)-, (2S)-, (2R)-1-(5-quinolyloxy)-3-alkylamino-2-propanols (10-21) and (2R,2S)-1-(5-isoquinolyloxy)-3-alkylamino-2-propanols (24-27) is described. beta 1-Adrenolytic (atria) structure-activity correlations indicated the N-alkyl substituent was a determinant of activity where the relative potency order was i-Pr and t-Bu > cyclohexyl. Compounds having the (2S)-configuration were more potent than the corresponding (2R)-analogs. The position of the heteroaryl nitrogen atom influences potency since the quinolyl analogs were ten-fold more active than the corresponding isoquinolyl isomers. All compounds in both the quinolyl and isoquinolyl series exhibited weak beta 2-adrenolytic activity (trachea), and high beta 1/beta 2 selectivity ratios when the N-alkyl substituent was i-Pr or t-Bu. The quinolyl ring system is an excellent bioisostere for the aryl moiety in aryloxypropanolamines since (2R,2S)- or (2S)-1-(5-quinolyloxy)-3-isopropyl (or t-Bu) amino-2-propanols exhibit very potent and highly cardioselective beta 1-adrenergic antagonist activities.


Antihypertensive therapy in experimental diabetes.

Systemic hypertension is an important risk factor for the progression of diabetic glomerular disease. Recognition of this detrimental aspect has prompted intensive investigation into the mechanisms by which systemic hypertension promotes diabetic glomerulopathy, as well as into potential benefits of antihypertensive therapy. Studies in diabetic rats, both normotensive and hypertensive, have established that certain antihypertensive regimens effectively slow the development of albuminuria and glomerular sclerosis. Most consistently effective have been angiotensin-converting enzyme inhibitors, which may act to protect the kidney by several different mechanisms. Other antihypertensive regimens have been less consistent.


Influence of preoperative dexamethasone therapy on proliferating cell nuclear antigen (PCNA) expression in comparison to other parameters in meningiomas.

We conducted a trial in 42 benign and malignant meningiomas to assess a possible influence of preoperative dexamethasone therapy on mitotic index, labelling indices of proliferating cell nuclear antigen (PCNA), progesterone receptor, epidermal growth factor receptor (EGF-R), c-erbB-2 oncoprotein, cathepsin D, gamma-gamma enolase as well as the mean number of silver-stained nucleolar organizer region-associated proteins (AgNORs). Tumors with preceding dexamethasone therapy for more than 1 day display significantly less immunohistochemical staining for PCNA. A correlation between the labelling index of PCNA and the degree of malignancy could not be identified. There was no significant effect of preoperative dexamethasone therapy on the other parameters. Our data suggest that dexamethasone may selectively inhibit the expression of PCNA in the G1/S-phase of the cell cycle. Thus, we emphasize the necessity to heed factors, e.g. dexamethasone, which may affect the expression of proliferating markers.


Morphometric changes in GH-immunoreactive adenohypophyseal cells induced by intraventricular administration of colchicine to adult rats.

In order to elucidate whether the gender differences observed in the somatotropic cells of adult rats are mediated by hypothalamic neuropeptides, a morphometric analysis was made of the GH-immunoreactive cells of adult rats treated intraventricularly with colchicine. The morphometric and morphological findings obtained were correlated to the basal serum levels of GH at the time of sacrifice. Treatment with colchicine was seen to increase serum GH levels; this increase was accompanied by an increase in the intensity of the reaction of the GH-cells and, morphometrically, an increase in their size due to an increase in the nuclear area, but with no significant changes in the cytoplasmic area. The results suggest that in the absence of somatostatin and GRF the basal release of GH is elevated in a similar fashion in both sexes, in turn suggesting that gonadal steroids might act at hypothalamic level on the release of somatostatin and, indirectly, on the intracellular pool of GH and hormonal secretion.


Immunohistochemical determination of P-glycoprotein in a rat mammary tumour treated with tamoxifen.

The effect of treatment with the antiestrogen Tamoxifen on the distribution and localization of P-glycoprotein was determined by immunohistochemistry in a rat mammary tumour model, the R3230AC. Both in the untreated and the treated tumours, P-glycoprotein is unevenly expressed with numerous negative tumour cells and located predominantly in the cytoplasmic membranes. Administration of Tamoxifen significantly lowers P-glycoprotein content of the tumour studied.


Localization of serotonin, cholecystokinin and somatostatin immunoreactivity in the lower respiratory tract of embryonic, foetal and postnatal sheep.

The lower respiratory tract of the sheep was studied by light-microscopical immunocytochemistry for serotonin, cholecystokinin, somatostatin, bombesin and calcitonin during different periods of lung development; embryonic, foetal and postnatal. At embryonic period only intraepithelial serotonin-containing cells as solitary neuroendocrine cells (NEC) and neuroepithelial bodies (NEB) were found. At foetal stages, immunoreactive cells to serotonin, cholecystokinin and somatostatin were observed in airway epithelium, as solitary NEC and NEBs, and in autonomic intrapulmonary ganglia as single or clusters of small intensely-fluorescent (SIF) cells. In postnatal sheep, serotonin- and cholecystokinin-containing cells were found within airway mucosa as solitary NECs and NEBs. No immunoreactive cells were observed with antiserum to bombesin and calcitonin. Quantitative studies showed that serotonin was the predominant substance, and that solitary neuroendocrine cells were more numerous in distal conducting airways and at foetal stages.


Neisserial surface variation: how and why?

Neisseria gonorrhoeae exhibits striking variability in several of its surface components (pili, Opa proteins and lipooligosaccharide) in vivo and in vitro. Such flagrant variation of this mucosal pathogen's surface components contrasts sharply with changes in single surface components of blood-borne trypanosomes and borreliae. Despite these differences, similar molecular events are sometimes involved.


[The serotonin- and dopaminergic mechanisms in the action of 1-pyrimidinyl piperazine derivatives].

It has been established in experiments on spinal ganglia neurons of rats that 1-pyrimidinyl-piperazine derivatives show the properties of partial agonists of 5-HT1A-receptors. Some of them were discovered to be capable of blocking D2-dopamine receptors. Comparison of neuronal and behavioral activity of the substances tested has demonstrated that their anxiolytic activity detectable under the conditions of the conflict situation method significantly correlates with 5-HT1A-mimetic and anti-dopamine activity. The latter one correlates well with the influence of the substances tested on the time of immobilization in the forced swimming test.


Effect of sulphasalazine on antibody response to oral antigen.

Cholera toxin was orally administered to mice concurrently receiving sulphasalazine (SASP) dissolved in L-lysine, or a control substance (L-lysine alone). Circulating antibodies to cholera toxin of IgM, IgG and IgA class were determined by direct ELISA at day 7, 14, 21 and 28. Although both groups made a significant antibody response to the antigen, mice receiving SASP tended to produce lower levels. These were significant for IgA on day 21 (P = 0.013), and for days 7-28 (P = 0.009), and 14-28 (P = 0.007). Overall, considering all antibody classes together from day 7 to 28, there was a significant effect in the SASP treated group (P = < 0.04). It appears that SASP exerts a mild immunomodulatory effect on the mucosal immune system. Further work is obviously required to substantiate these findings. The effect on the gut mucosal immune system of a drug known to ameliorate rheumatoid arthritis may offer an insight into the aetiopathogenesis of this disease.


[The dose-dependent effect of mezaton and fetanol on the glucose level of the blood].

Experiments with mice and rabbits have revealed that mezaton and fetanol in a dose of 0.1 mg/kg are able to cause heterodirectional changes in blood glucose levels. When their dose is increased to 1 and 10 mg/kg, the two agents result in hyperglycemia.


[The effect of narcotic analgesics on nociceptive reactions of a visceral and somatic nature].

The experiments on conscious cats showed that buprenorphine given in 0.0125 and 0.025 mg/kg selectively inhibited motor, emotional and hemodynamic manifestations of pain induced by stimulation of the splanchnic nerve. The analgetic effect of morphine, tramal, pentazocine and butorphanol was equally demonstrated on visceral and somatic pain models, but the drugs elevated the baseline blood pressure and produced no (morphine, pentazocine) or moderate (tramal, butorphanol) antihypertensive effects in various nociceptive stimuli.


Genetically epilepsy-prone rats have increased brain regional activity of an enzyme which liberates glutamate from N-acetyl-aspartyl-glutamate.

N-Acetylated-alpha-linked acidic dipeptidase (NAALADase) is a membrane-bound peptidase which hydrolyzes the endogenous neuropeptide N-acetylaspartylglutamate (NAAG) to N-acetylaspartate (NAA) and the excitatory amino acid, glutamate (Glu). Although there is evidence that NAAG might be a neurotransmitter, this dipeptide could also function as a precursor form of Glu, which is liberated by the dipeptidase. We found that the activity of this NAAG hydrolyzing enzyme in genetically epilepsy-prone rats was 11-26% greater than control in brain regions, including the amygdala, hippocampus and cerebellum, as well as the pyriform, entorhinal and frontal cortices. This is consistent with possible increased availability of Glu in certain CNS synapses in these rats, which are reported to have increased susceptibility to audiogenically, electrically and chemically induced convulsions.


Consequences of an intrastriatal injection of kainic acid on the dopaminergic neuronal and vesicular uptake systems.

Intrastriatal kainic acid injection destroys the neurons originating from the striatum, including those on which terminals of the nigro- and meso-striatal dopaminergic neurons project. We have studied at various times the consequences of this lesion on dopamine metabolism and on the neuronal and vesicular transporters. Two and 15 days after kainic acid injection, whereas dopamine turnover was increased and the dopamine content unchanged, there was no modification in the binding of [3H]GBR 12783, a marker of the neuronal uptake complex, but the binding of [3H]dihydrotetrabenazine, a marker of the vesicular transporter, was significantly decreased. At later times (30 and 60 days) when the dopamine turnover was decreased as well as the dopamine content, the binding of [3H]dihydrotetrabenazine was more dramatically decreased (about 30% of controls) than that of [3H]GBR 12783. In addition autoradiography showed an increase in the density of [3H]dihydrotetrabenazine binding sites in the substantia nigra. Thus it appears that the long-term (60 days) repercussions of a kainic acid lesion affect simultaneously the dopamine turnover (which is decreased) and the vesicular transporter whereas the dopamine uptake complex is little affected.


Effects of ethanol on Na(+)-dependent amino acid uptake: dependence on rat age and Na+, K(+)-ATPase activity.

Acute effects of ethanol on Na(+)-dependent transport of gamma-aminobutyric acid (GABA) and glutamic acid (GLU) were investigated in crude synaptosomal preparations from rat cerebral cortex. In experiments with 30-40-day-old (peripubertal) rats, the overall dose responses of the GABA and GLU transport systems to ethanol were biphasic. Stimulation was observed at ethanol concentrations (40-160 mM) relevant to intoxication. Inhibition was observed at higher concentrations of ethanol. The stimulatory phase of the dose response was not observed in 60-100-day-old (adult) rats. In preparations from peripubertal rats, other alcohols also had biphasic dose response curves with stimulation at low alcohol concentrations. The relative efficacy of the different alcohols appeared to correlate with the relative membrane-buffer partition coefficient. In synaptosomal membrane vesicles, where artificial ion concentration gradients rather than Na+,K(+)-ATPase activity provide the driving force for uptake, ethanol did not stimulate GABA uptake. In direct measures of Na+,K(+)-ATPase activity, both Rb+ uptake and ATP hydrolysis were enhanced by 80 mM ethanol. We conclude that stimulation of Na(+)-dependent uptake of amino acids by ethanol was secondary to enhanced Na+,K(+)-ATPase activity and may be associated with a specific developmental stage in the rat.


A voltage-clamp analysis of NMDA-induced responses on dopaminergic neurons of the rat substantia nigra zona compacta and ventral tegmental area.

The effects of NMDA-receptor activation on dopaminergic neurons of the rat substantia nigra zona compacta and ventral tegmental area were studied by using in vitro intracellular electrophysiological recordings (current and voltage-clamp). NMDA depolarized the membrane and increased the firing activity. A voltage-dependent inward current and a reduction of the apparent input conductance were observed in voltage-clamp experiments. Interestingly, the peak amplitude of the inward current occurred at approximately -60 mV. The NMDA-induced responses were reduced by the application of DL-2-amino-5-phosphonovaleric acid (APV). The NMDA-induced current was unaffected by potassium channel blockers, was present in low-sodium solutions or in solutions treated with TTX; but was reduced or blocked in low-calcium solutions containing cobalt. In addition, no reduction of the apparent input conductance was observed either in the solutions without magnesium or in those with low-sodium. Our data indicate that the activation of NMDA receptors produces a powerful excitatory stimulus on the dopaminergic neurons of the ventral mesencephalon and this may be primarily the result of a voltage-dependent influx of calcium ions. The degeneration of the dopaminergic cells after application of neurotoxins may be explained by their peculiar response to NMDA.


Spermine facilitates the generation of long-term potentiation of evoked potential in the dentate gyrus of anesthetized rats.

The effects of the polyamines, spermine, spermidine and putrescine, on long-term potentiation (LTP) of evoked potential were investigated in the dentate gyrus of anesthetized rats. Injection of 5 nmol spermine into the lateral ventricle did not influence the basal amplitude of the population spike, but significantly enhanced the potentiation induced by subthreshold tetanic stimulation (20 pulses at 60 Hz). The effect of spermine resulted in facilitation of LTP generation. Injection of the same dose of spermidine or putrescine affected neither the basal response nor the potentiation induced by subthreshold tetanus at all, indicating that the LTP-facilitating effect is specific to spermine. Furthermore, the LTP-facilitating effect of spermine was dose-dependent in the range of 0.5-50 nmol. When 5 nmol ifenprodil, an antagonist at the polyamine site of the NMDA receptor channel complex, was concomitantly injected, spermine could not facilitate the generation of LTP. Since injection of ifenprodil alone did not influence the generation of LTP, it is probable that ifenprodil specifically blocks the effect of spermine. These results suggest that spermine facilitates the generation of hippocampal LTP, probably through an ifenprodil-sensitive polyamine site associated with the NMDA receptor.


Potent antinociceptive effects of clonidine systemically administered in an experimental model of clinical pain, the arthritic rat.

The effects of various doses of the alpha-2 adrenoceptor agonist clonidine administered systemically (30, 50 and 100 micrograms/kg i.v.), were investigated on the vocalization threshold to paw pressure in normal rats and in rats with Freund's adjuvant-induced arthritis. Previous results have suggested that there is an increase in the activity of the bulbospinal noradrenergic systems in these arthritic animals. In the present study, clonidine led to significant antinociceptive effects in both groups of rats. Clonidine was found to be highly effective in arthritic animals, even at the lower concentration: the elevation in threshold produced by 30 micrograms/kg i.v. was 160% in arthritic vs. 124% in normal rats. The effects of clonidine were prevented dose-dependently by pretreatment with yohimbine or idazoxan 250 to 1000 micrograms/kg i.v., in the two groups of rats, indicating clearly that the dose-dependent effects of i.v. clonidine are mediated by alpha-2 adrenoceptors.


Characterization of phencyclidine-induced effects on neuropeptide Y systems in the rat caudate-putamen.

Multiple administrations of the psychotomimetic drug, phencyclidine-HCI (PCP), decreased striatal neuropeptide Y-like immunoreactivity (NPY-LI) levels in a dose-dependent manner. Single or multiple PCP administrations decreased striatal NPY levels after 10-12 h; levels returned to control 24 h after a single dose or 58 h after multiple doses. In contrast, no significant changes were seen in nigral NPY levels with either acute or multiple-dose PCP treatments. The role of monoamine, sigma or opioid receptors in PCP-induced striatal NPY changes was evaluated. When administered alone, the alpha 1-adrenergic antagonist, prazosin, the sigma antagonist, BMY 14802, and the dopamine D2 antagonist, sulpiride decreased striatal NPY levels; however, only prazosin and the dopamine D1 antagonist, SCH 23390, significantly attenuated PCP-induced changes. Administration of the gamma-aminobutyric acid transaminase (GABA-T) inhibitors, amino-oxyacetic acid (AOAA) or gamma-vinyl-GABA (GVG, vigabatrin, MDL 71,754) alone had no effect on striatal NPY-LI levels while administration of these indirect GABA agonists prior to or concurrently with PCP treatment completely blocked PCP-induced changes in striatal NPY-LI levels. The effect of the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, on striatal NPY-LI content resembled that of PCP and was also blocked by the two indirect GABA agonists. These data suggest that NPY systems are modulated by glutamatergic activity (specifically by the NMDA receptor) and that the interaction between these two transmitter systems is mediated by GABAergic mechanisms.


Chromosome 21 rearrangement in acute biphenotypic leukemia.

A patient with myelodysplastic syndrome (MDS) and a 47,XY,+21 karyotype at diagnosis, was documented to have a clonal chromosome 21 rearrangement, i(21q), four months before transformation to acute biphenotypic leukemia. For 4 months after transformation, isochromosome 21 persisted while the patient was receiving treatment with zidovudine. Vitamin D3 was added to zidovudine for an additional month, during which time the trisomy 21 clone reappeared as the predominant cell population. The unique aspects of this patient are the atypical evolution of chromosome 21, the transformation to biphenotypic leukemia, and the occurrence of i(21q) associated with biphenotypic leukemia evolving from an MDS.


Allelic loss from chromosome 11 in parathyroid tumors.

Parathyroid tumors may occur in a sporadic fashion or, more rarely, as part of a familial syndrome (such as familial multiple endocrine neoplasia type I). The MENI gene has been mapped by linkage analysis to chromosome 11 at band q11-q13, and presumably acts as a tumor suppressor gene. In the present study, which is an extension of our previous studies, we examined 41 parathyroid tumors from patients with familial multiple endocrine neoplasia type I and 61 sporadic parathyroid tumors with markers on chromosome 11, to assess the extent of allelic loss in those tumors. Twenty-four of the MENI-associated tumors (58%) and 16 of the sporadic parathyroid tumors (26%) displayed allelic loss from chromosome 11. The region of overlap of the allelic losses in the MENI-associated tumors enables us to place the MENI gene between PGA centromerically and INT2 telomerically, a region spanning about 7.5 cM. Taken together with locus ordering by linkage analysis, this clearly localizes the MENI gene telomeric to the PGA locus. Our inability to detect allelic loss on chromosome 11 in some parathyroid tumors suggests the existence of other genes involved in the development and/or progression of this subgroup of presumably monoclonal tumors; or that localized events involving the 11q tumor suppressor gene have occurred in some parathyroid tumors whose detection is beyond the sensitivity of our analysis; or that at least some of the specimens analyzed were in fact primarily hyperplastic parathyroid tissue.


Histogenetic analysis of ovarian germ cell tumors by DNA fingerprinting.

The histogenesis of ovarian germ cell tumors (11 mature teratomas, three malignant transformations of mature teratomas, two immature teratomas, and four dysgerminomas) was investigated genetically using minisatellite DNA probes 33.15 and 33.6 for person-specific restriction fragment length polymorphism (DNA fingerprint) analysis. The DNA fingerprints of six ovarian teratomas were identical with those of mononuclear cells from each host, while some polymorphic bands observed in the host mononuclear cells were lost in the DNA fingerprints of the other five cases. The cases of malignant transformation of mature teratoma and immature teratoma showed that some polymorphic bands of DNA fingerprints from the host mononuclear cells were absent in the tumor tissues. In four cases with dysgerminomas, the DNA fingerprints of tumors were completely identical with those of the respective host mononuclear cells. The present results suggest that mature cystic teratomas of the ovary arise from germ cells arrested at various stages of meiosis, while immature teratomas are derived from postmeiotic germ cells. Malignant transformation may occur exclusively in the mature teratomas arising from postmeiotic germ cells. Dysgerminomas develop from premeiotic oogonia (primordial germ cells). Thus, DNA fingerprints are a useful and sensitive tool for identifying the pathogenesis of germ cell tumours.


In vitro cytotoxic targeting by human mononuclear cells and bispecific antibody 2B1, recognizing c-erbB-2 protooncogene product and Fc gamma receptor III.

Bispecific murine monoclonal antibody 2B1, possessing dual specificity for the human c-erbB-2 protooncogene product and human Fc gamma receptor III (CD16) was evaluated for the ability to promote specific lysis of c-erbB-2-positive tumor cells in vitro. In short-term 51Cr release assays with human mononuclear cells as effectors and SK-Br-3 human breast cancer cells as targets, neither parental antibody of 2B1 mediated significant specific lysis, but bispecific antibody was as active as a chemical heteroconjugate, with 5 ng/ml of 2B1 causing half-maximal lysis at an effector/target ratio of 20:1 and 2 ng/ml 2B1 causing half-maximal lysis at an E/T ratio of 40:1. The cytotoxic targeting activity of 2B1 F(ab')2 fragment was the same as that of whole bispecific antibody, and the activity of whole 2B1 was not reduced when assays were performed in 100% autologous human serum, indicating that 2B1 binds effector cells through the CD16-binding site derived from parental antibody 3G8 rather than through its Fc portion. Variable inhibition of 2B1-mediated lysis was observed when autologous polymorphonuclear leukocytes from different donors were added to mononuclear effector cells at a 2:1 ratio; this inhibition was overcome at higher antibody concentration. 2B1 bispecific monoclonal antibody was also able to mediate targeted cytolysis using whole human blood as a source of effector cells or using effector or target cells derived from ovarian cancer patients.


Expression and functional activity of P-glycoprotein in cultured cerebral capillary endothelial cells.

Analysis of a panel of endothelial cells passaged between 5 and 25 times and derived from various organs and species demonstrated that murine and porcine cerebral capillary endothelial cells actively excluded the fluorescent dye rhodamine 123, a substrate of P-glycoprotein. In addition, rhodamine 123 accumulation could be enhanced by the multidrug resistance chemosensitizer verapamil, known to reduce P-glycoprotein-mediated drug efflux. Cloned murine and porcine cerebral capillary endothelial cells were immunoreactive with the C219 monoclonal antibody to P-glycoprotein, and a C219 epitope-specific blocking peptide could abolish staining. The antiproliferative and cytotoxic effects of vincristine, but not cis-platinum(II) diamminedichloride, were increased by the addition of either verapamil or cyclosporin A to brain endothelial cell cultures in a 72-h assay, as determined by [3H]thymidine incorporation and total protein measurement. Cyclosporin A was a more effective reversal agent than verapamil. Thus, a P-glycoprotein isoform may be constitutively expressed in brain endothelial cells in vitro and supports the available data on in situ immunohistochemical staining of P-glycoprotein at the blood-brain barrier. In addition, these findings may indicate that one function of P-glycoprotein in vivo at the blood-brain barrier is the exclusion of xenobiotics from central nervous system tissues.


Homeotic transformations in the mouse induced by overexpression of a human Hox3.3 transgene.

A permanent transgenic mouse line was generated carrying 40 copies of the human Hox3.3 gene. The resulting mice express large amounts of Hox3.3 protein in posterior regions of the embryo where this homeodomain protein is normally not expressed. The transgene causes homeotic transformations of the skeleton, in particular the appearance of an extra pair of ribs in the lumbar region, transformation of the shape of posterior ribs into that of more anterior ones, and the joining of an additional pair of ribs to the sternum. The phenotype of this line resembles that obtained by the targeted loss-of-function mutation of Hox3.1 (Le Mouellic et al., 1992). In transient assays, the human Hox3.3 transgene leads to the formation of additional ribs in more posterior vertebrae as well.


Altering the boundaries of Hox3.1 expression: evidence for antipodal gene regulation.

To investigate the function of region-specific patterns of mouse homeobox gene expression during embryogenesis, we programmed a minimal change in the distribution of Hox3.1 transcripts along the anteroposterior body axis in transgenic mice. Regulatory sequences from Hox1.4, a gene normally expressed more anteriorly than Hox3.1, were chosen to direct expression of a Hox3.1 transgene. Offspring of independent transgenic lines expressed the transgene more anteriorly than the Hox3.1 gene. Rather than predicted posterior transformations, we observed anterior transformations of vertebrae in newborn mice. Transgenic mice also developed profound gastrointestinal tissue malformations, which may provide a molecular explanation for human developmental disorders often involving these same two regions. Paradoxically, vertebral transformations in the transgenic mice were strikingly similar to those reported in mice homozygous for a null mutation of the Hox3.1 gene. This observation suggests that Hox genes may be regulated antipodally, with over- or underexpression resulting in similar phenotypes.


Doxorubicin sensitivity pattern in a panel of small-cell lung-cancer cell lines: correlation to etoposide and vincristine sensitivity and inverse correlation to carmustine sensitivity.

The aim of our investigations is to evaluate whether the sensitivity patterns of small-cell lung-cancer (SCLC) cell lines in vitro can be used in evaluating new drugs and in selecting drugs for the optimization of combination therapy. In our attempts to obtain a panel of cell lines demonstrating differential patterns in sensitivity, we have developed three SCLC lines exhibiting different types of multidrug resistance (MDR). In the present investigations we compared the sensitivity patterns shown by five wild-type SCLC lines and three MDR lines in response to six different types of drugs: doxorubicin, cytarabine, carmustine, cisplatin, vincristine, and etoposide. In the wild-type SCLC cell lines, the range of variation in sensitivity to all drugs was within a factor of 10. Cell lines showing low sensitivity to doxorubicin also exhibited low sensitivity to etoposide and vincristine, and vice versa. In contrast, the pattern of sensitivity to carmustine was almost the opposite of that to doxorubicin. A tendency to an inverse relationship between doxorubicin and carmustine sensitivity was also observed when doxorubicin sensitivity was reduced in near stationary cells and in cells exposed to the metabolic inhibitor 2-deoxy-D-glucose. In agreement with the pattern observed for the wild-type lines, all of the MDR sublines demonstrated collateral sensitivity to carmustine. As to cytarabine, the wild-type lines expressed a sensitivity pattern similar to that shown in response to doxorubicin. Interestingly, the opposite pattern was found in the MDR lines, as all three demonstrated cytarabine hypersensitivity. The combination of alkylating agents and "MDR" drugs are of proven clinical benefit in the treatment of solid tumors, as is the combination of anthracycline and cytarabine in acute myeloid leukemia. The experimentally derived sensitivity data on cytarabine, alkylating agents, and MDR drugs (i.e., etoposide, doxorubicin, vincristine) thus resemble the clinical experience with these drugs, and we conclude that the use of a clonogenic assay on the described panel of SCLC cell lines can give valuable information for the selection of agents for combination therapy.


Missense mutations of the tissue-nonspecific alkaline phosphatase gene in hypophosphatasia.

Hypophosphatasia is an inborn error of metabolism that is characterized clinically by defective bone mineralization and biochemically by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) in serum and in tissues. Clinical severity is extremely variable, ranging from death in utero to pathologic fractures first presenting in adulthood. Severe forms of the disease are inherited in an autosomal recessive fashion; the modes of transmission of mild forms are uncertain. Deficiency of TNSALP activity in this condition suggests that mutations in the TNSALP "candidate" gene are the primary defects. This hypothesis was supported in 1988 by the demonstration, in one inbred infant, that an identical missense mutation in both alleles of the gene encoding TNSALP caused lethal hypophosphatasia. Here we summarize the work leading to that discovery and discuss the recent identification of additional missense mutations in the TNSALP gene associated with the entire clinical spectrum of hypophosphatasia.


CD4-independent binding of HIV-1 to the B lymphocyte receptor CR2 (CD21) in the presence of complement and antibody.

Complement and antibody contribute to infection-enhancement and possible expanded cellular tropism of HIV-1 in vitro through a process requiring complement receptors. Until now, however, the ability of HIV-1 to bind complement receptors has not been documented or characterized. We investigated whether antibody and complement permitted HIV-1 to bind to the B lymphocyte receptor, CR2 (CD21), in an effort to learn more about infection-enhancement, and also because CR2 can mediate B cell proliferation and antigen localization in lymphoid organs in other systems. HIV-1 incubated with antibody and fresh human serum as a source of complement bound approximately 10-fold greater to cells expressing CR2 than to HIV-1-permissive cells lacking this receptor. A similar effect was observed using cells which expressed CR2 but no CD4. This binding was minimal in heat-inactivated and C3-deficient sera, and was significantly reduced by the anti-CR2 MoAb, OKB7, but not by the anti-CD4 MoAb, OKT4a. Thus, complement and antibody acted in concert to facilitate the binding of HIV-1 to CR2 independently of CD4. CD4-independent binding of HIV-1 to CR2 was not sufficient to produce infection in Raji-3 cells. Titres of antibodies mediating CR2 binding correlated with antibody titres as measured by immunofluorescence (P < 0.01) and infection-enhancement (P < 0.05) but were discordant with titres of neutralizing antibodies, a result consistent with the utilization of CR2 for enhanced infection of cells. The ability of complement and antibody to facilitate the binding of HIV-1 to CR2 in the absence of CD4 provides new insights into mechanisms of HIV-1-induced immunopathogenesis and infection-enhancement.


Haemorrhage-induced alterations in function and cytokine production of T cells and T cell subpopulations.

Haemorrhage produces alterations in macrophage, T and B cell function. In order to better define the mechanism for the effects of blood loss on immune response, we examined function of and cytokine production by purified T cells, CD4+ and CD8+ subpopulations after blood loss. Whereas T and CD4+ cells from control, unhaemorrhaged animals produced no alteration in proliferation when added to cultures of mitogen-stimulated splenocytes from normal mice, proliferation was decreased when T or CD4+ cells from haemorrhaged mice were included. The addition of CD8+ cells from haemorrhaged animals to mitogen-stimulated cultures reduced proliferation by approximately 50% more than that found when CD8+ cells from control, unhaemorrhaged animals were included. Supernatants of mitogen-stimulated splenocytes from haemorrhaged mice contained significantly less IL-2 and interferon-gamma (IFN-gamma) than did those from control, unhaemorrhaged mice. CD4+ populations from haemorrhaged mice produced significantly more IL-10, and significantly less IFN-gamma, than did CD4+ cells from control, unhaemorrhaged mice. There were no significant differences in IL-2, IL-4, IL-10 or IFN-gamma production by CD8+ cells from haemorrhaged or control mice. The present experiments demonstrate that haemorrhage affects both CD4+ and CD8+ T cell subsets. In particular, haemorrhage appeared to activate CD4+, Th2 cells, with concomitant suppression of the Th1 subpopulation. These results provide a mechanism which may contribute to the alterations in cytokine production previously described to occur following blood loss.


Expression and gene transcript of Fc receptors for IgG, HLA class II antigens and Langerhans cells in human cervico-vaginal epithelium.

The mechanism of transmission of HIV from the male to the female genital tract or in the reverse order is not clear. CD4 glycoprotein is the receptor for HIV and Langerhans cells and the related dendritic cells could play a role in the initial transmission of HIV. Fc receptors (FcR) for IgG might be involved in antibody-mediated binding of HIV. We carried out an immunohistological study of normal human cervical and vaginal epithelia for the presence of CD4 glycoprotein, Langerhans cells and FcR to IgG. CD4+ glycoprotein was not found in the vaginal or cervical epithelium, with the exception of a few endocervical epithelial cells. A small number of CD4+ mononuclear cells were found in the endocervical epithelium of a third of the specimens but a large number of CD4+ cells was found in the submucosa of most of the cervical and vaginal specimens. Langerhans cells expressing CD4, HLA class II, Fc gamma R2 and Fc gamma R3 were detected in most vaginal, ectocervical and transformation zone epithelia and in 9/14 endocervical tissues. Fc gamma R3 was detected in about two-thirds of the columnar endocervical epithelium and the transformation zone. A smaller number of specimens expressed Fc gamma R2 in these epithelia, but Fc gamma R1 was not detected. We then demonstrated mRNA for Fc gamma R3 in the columnar endocervical epithelial cells and transformation zone by in situ hybridization, using a CD16-RNA probe. Fc gamma R3 and Fc gamma R2 gene transcripts were also found in fetal cervical tissue by applying the polymerase chain reaction to amplify portions of the Fc gamma R3 and Fc gamma R2 coding sequences in cDNA prepared from fetal RNA. HLA-DR was found in the endocervical cells, transformation zone and in Langerhans cells of all specimens. The presence of Langerhans cells, Fc gamma receptors and HLA class II antigen offers three potential mechanisms for cervico-vaginal HIV transmission: (i) direct HIV infection of Langerhans cells, (ii) binding of HIV antibody complexes to cervical epithelial Fc gamma receptors and (iii) binding of HIV infected CD4+ cells to cervical HLA class II antigen which may infect these or the adjacent CD4+ cells.


New insights and technologies in brain grafting.

Significant progress has been made in the field of brain grafting over the last 15 years. Neurosurgeons have been involved directly in the preclinical and clinical efforts in this fascinating and promising field, along with their neuroscience colleagues. Through a better understanding of the complex mechanisms involved in response to transplants in the brain, new technologies and experimental strategies are being developed to improve the safety and efficacy of these procedures. The time is right for carrying out appropriate preclinical studies in rodents and nonhuman primates to answer one of the most basic questions: Is a tissue graft necessary for behavior improvement in degenerative diseases such as PD, HD, or AD? With available tools and technology and an open mind to new ideas, brain grafting has a tremendous potential in the neurosurgeon's armamentarium, both today and in the future.


Accessory molecules expressed on the peripheral blood or synovial fluid T lymphocytes from patients with Sjögren's syndrome or rheumatoid arthritis.

We previously demonstrated that the cells expressed by activation antigens were increased in several T cell subsets from the peripheral blood (PB) and joint fluid (JF) of patients with Sjögren's syndrome (SS) or rheumatoid arthritis (RA). In the present report, we further determined by three-color flow cytometry the homing receptors (Leu8) for peripheral lymph nodes expressed on naive (CD45RA+) or memory (CD45RA-) CD4+ cells and on the two subsets of CD8+ cells (CD11b+ and CD11b-) in the PB and JF from SS and RA patients. In addition, the activation antigens (HLA-DR) and two adhesion molecules, including the alpha-chain of the leukocyte function associated antigen-1 (LFA 1 alpha: CD11a) and its ligand (intercellular adhesion molecule-1: ICAM-1: CD54) expressed on T cells, were compared. We found that CD45RA-CD4+ cells were markedly increased in JF, while CD45RA+CD4+ cells were almost absent. Leu8+ cells were decreased in both CD45RA-CD4+ and CD8+CD11b-cells (cytotoxic T cells) in the JF, and were also decreased in the CD8+CD11b-subset in the patients' PB. Furthermore, activated (DR+) T cells were markedly increased in JF, and the cells expressed more adhesion molecules in both the PB and JF from patients, compared with the DR-T cells. The DR+ T cells therefore are considered to be memory T cells, which are more efficient for cell-to-cell interactions. These observations also suggest that the Leu8- and DR+ T cells with increased adhesion molecules might preferentially migrate into inflammatory tissues, and that naive T cells are being further converted to to memory T cells by in vivo stimulation within the tissues.


Demonstration of calf abnormalities by magnetic resonance imaging in polyarteritis nodosa.

A 36-year-old Caucasian woman presenting with persisting painful calves after a flu-like illness was diagnosed as having polyarteritis nodosa. Magnetic resonance imaging of the lower legs showed abnormal signal intensity of the outer muscle groups with sparing of the central located muscles. The good clinical response to oral prednisone was supported by improvement of MRI.


Allele loss on chromosome 11p is associated with poor survival in ovarian cancer.

RFLP (restriction fragment length polymorphism) analysis in 46 ovarian tumour and paired blood samples shows that 33 per cent (5/15) of informative carcinomas had loss of heterozygosity (LOH) at 11p15.4 (Calcitonin locus) and 18% (4/22) had LOH at 11p15.5 (Ha-ras). No LOH was detected in five borderline and four benign ovarian tumours. Analysis of survival in the carcinoma group (37 patients) showed a significantly poorer survival in patients whose tumours showed LOH at either or both of these 11p loci (chi 2 = 7.771, p = 0.005). This suggests the presence of a tumour suppressor gene (tsg) on the short arm of chromosome 11 whose loss is associated with particularly aggressive disease.


Reversible shutdown of replicon initiation by transient hypoxia in Ehrlich ascites cells. Dependence of initiation on short-lived protein.

The O2-dependent regulation of replication in Ehrlich ascites cells, characterized by a reversible shutdown of replicon initiation during hypoxia, was scrutinized with respect to the involvement of gene expression. Synchronous and asynchronous cells were subjected to transient hypoxia and examined for expression of selected 'late' growth-regulated mRNA and for the influence of inhibitors of transcription and translation on DNA replication. Irrespective of whether replicon initiation was suppressed by hypoxia or retriggered by reoxygenation, the levels of thymidine kinase mRNA and of proliferating cell-nuclear antigen/cyclin mRNA were as high as in untreated replicating cells. The level of histone H3.1 mRNA followed, with a distinct delay, the replicative activity of the cells governed by the imposed changes of pO2. The response of replication to inhibition of transcription and translation was virtually the same as to hypoxia, i.e. a selective suppression of replicon initiation. It was demonstrated that replicon initiation depends on one or several short-lived protein(s) (lifetime about 5 min) which is (are) formed under hypoxic conditions as well. The lifetime of the corresponding RNA message(s) is in the range of several hours. It is suggested that the expression of genes conditioning resting cells for DNA replication remains unaffected by hypoxia or by restoring the normal pO2. Hypoxic cell appear to rest in a state fully prepared for entering DNA replication, but a yet unknown event essential for replicon initiation is blocked. This event depends on a critical oxygen tension as well as on short-lived protein(s).


Temperature-gradient gel electrophoresis for the detection of polymorphic DNA and for quantitative polymerase chain reaction.

In order to detect mutations in a gene, either known mutations from human diseases or artificial ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarantees detection of mutations which might occur in every single position of the whole open reading frame (ORF). It will be shown that a combination of polymerase chain reaction (PCR) and temperature gradient gel electrophoresis (TOGE) fulfills these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated in dependence on the position of the mutation. The theoretical results were tested with the mutations known so far. The quantitative determination of the copy number of a specific DNA or RNA sequence in a biological specimen (quantitative PCR) can be performed precisely and easily by combining PCR and TGGE. The system uses a quantification strategy of a new type of internal standardization. TGGE is applied to separate homo- and heteroduplexes which correspond respectively to standard and template sequences. The accuracy of this quantification strategy is very high, with a variability of < 15%. In addition to quantification, PCR/TGGE detects PCR artifacts and template mutants.


Severe type III hyperlipoproteinemia associated with unusual apolipoprotein E1 phenotype and epsilon 1/'null' genotype.

A 60-year-old white male (KH) was diagnosed to suffer from severe type III hyperlipoproteinemia (HLP) and premature cardiovascular disease. Biochemical analysis revealed an unusual apolipoprotein (apo) E phenotype and genotype. All clinical characteristics of type III HLP were present in the patient. His very low density lipoprotein (VLDL) cholesterol to plasma triglyceride (TG) ratio was elevated at 0.97 without therapy which is unusually high (normal ratio about 0.18). By contrast his plasma apo E level was only moderately elevated (6.8 mg dl-1). The patient's apo E migrated in the apo E1 position on isoelectric focusing gels. Chemical modification with cysteamine and treatment with neuraminidase confirmed the presence of two cysteine residues in the patient's apo E and a normal sialylation pattern. Pedigree analysis suggested that the patient was a compound heterozygote with one apo epsilon 1 allele and another allele whose product did not appear in the plasma compartment ('null' allele). Direct sequencing of polymerase chain reaction (PCR) amplified segments of the apo E gene as well as restriction fragment length polymorphism (RFLP) analysis with the endonuclease Taq I identified an adenosine for guanosine (G-->A) exchange in the second base of codon 127 that is predictive for an Asp for Gly substitution in the encoded apo E amino acid sequence. This mutation is the structural basis for the apo E1 isoform identified upon isoelectric focusing. Five other family members are also carriers of the mutant apo epsilon 1 allele. Two of those were hyperlipidemic and exhibited biochemical characteristics of type III HLP. A second mutation, a deletion of a G in codon 31, is predictive for a reading frameshift that encodes for a premature stop in codon 60. Our inability to identify the product of a second apo E allele in the plasma of the patient and two other members of the KH family corresponds with the heterozygous presence of this mutation in the affected individuals. Both relatives (like the index case) had an increased VLDL cholesterol to plasma TG ratio, which indicates the presence of cholesterol-enriched VLDL particles. We propose that the single base deletion in the apo E gene which is the cause of a non-functional 'null' allele in addition to a probably dominant apo E1 (Gly127-->Asp, Arg158-->Cys) variant of late or incomplete penetrance are the primary genetic defects in this kindred leading to severe dysbetalipoproteinemia.


Ethacrynic acid-induced convulsions and brain neurotransmitters in mice.

Intracerebroventricular injection of ethacrynic acid (50% convulsive dose; 50 micrograms/mouse) accelerated the synthesis/turnover of 5-hydroxytryptamine (5-HT) but suppressed the synthesis of gamma-aminobutyric acid and acetylcholine in mouse brain. These effects were completely antagonized by pretreatment with a glutamate/N-methyl-D-aspartate antagonist, aminophosphonovaleric acid. In ethacrynic acid-induced convulsions, these neurotransmitter systems may be differentially modulated, probably through activation of glutaminergic neurons in the brain.


Simple repeats are not found more abundantly at G/C-rich regions.

Three hundred and fifty cosmids from chromosome 7, previously analyzed for the presence of rare-cutting restriction enzyme sites, were analyzed for the presence of microsatellite sequences [(CA)n or (CT)n repeats]. Of these, 147 cosmids were found to contain at least one (CA)n repeat unit and 51 cosmids contained at least one (CT)n repeat unit. No evidence was found for the prevalence of microsatellite repeat units in the vicinity of rare-cutter restriction enzyme sites.


Effect of 5-HT receptor and adrenoceptor antagonists on micturition in conscious cats.

Micturition was induced in awake cats by infusing saline into the bladder at a physiological filling rate. Methysergide, a serotonergic antagonist given intrathecally, decreased the volume at which micturition occurred. Phentolamine, a non-specific alpha-adrenoceptor antagonist, also decreased volume threshold. Prazosin, an alpha 1-adrenoceptor antagonist, was without effect on micturition. These results imply that 5-HT receptors and alpha 2-adrenoceptors may be inhibitory to micturition at a spinal level.


Microinjection of S-nitrosocysteine into the nucleus tractus solitarii of conscious rats decreases arterial pressure but L-glutamate does not.

Unilateral microinjection of L-glutamate into the nucleus tractus solitarii of conscious rats increased arterial pressure and caused bradycardia while microinjection of S-nitrosocysteine into the same site of these animals caused hypotension and bradycardia. The responses to S-nitrosocysteine were blocked by prior microinjection of methylene blue into the nucleus tractus solitarii. The bradycardia and fall in arterial pressure induced by S-nitrosocysteine resemble more the cardiovascular changes in response to activation of baroreceptor afferents than the bradycardia and increase in arterial pressure induced by microinjection of L-glutamate into the nucleus tractus solitarii of conscious rats.


Antagonism of ethanol withdrawal convulsions in Withdrawal Seizure Prone mice by diazepam and abecarnil.

Abecarnil, a beta-carboline acting at benzodiazepine receptors, has been shown to have anxiolytic and anticonvulsant properties in a number of models. It has reduced muscle relaxant and incoordinating effects in comparison to diazepam. Given the wide clinical application of diazepam to prevent alcohol withdrawal seizures, a genetic animal model was employed to compare abecarnil with diazepam for its anti-withdrawal effects. Withdrawal Seizure Prone (WSP) mice, genetically selected to develop severe handling-induced convulsions after withdrawal from chronic ethanol treatment, were exposed to ethanol vapor for 24 h. WSP mice given doses of abecarnil or diazepam at the peak of withdrawal had significantly reduced handling-induced convulsion scores. While abecarnil was slightly more potent than diazepam, its effects were shorter-lasting. Similar results were seen in an experiment where withdrawal handling-induced convulsions were assessed after a single high-dose ethanol injection. Abecarnil and diazepam also reduced the smaller handling-induced convulsion scores seen in naive WSP mice. Single doses of abecarnil or diazepam did not lead to a rebound elevation of handling-induced convulsion scores suggestive of a withdrawal reaction.


Adenylate cyclase from sea urchin eggs is positively and negatively regulated by D-1 and D-2 dopamine receptors.

The adenylate cyclase present in membranes prepared from sea urchin eggs is sensitive to dopamine stimulation. The receptor sites coupled to sea urchin adenylate cyclase were characterized by means of specific agonists and antagonists. The D-1 dopamine agonist SKF-38393 was able to stimulate enzyme activity, while the two D-1 dopamine antagonists, SCH-23390 and SKF-83566, suppressed the stimulatory effect of dopamine. In addition, the D-2 dopamine agonists, PPHT and metergoline, brought about a dose-dependent inhibition of dopamine-stimulated adenylate cyclase activity. These data show that: (i) in sea urchin eggs adenylate cyclase is regulated by dopamine receptors; (ii) these receptors share characteristics with D-1 and D-2 dopamine receptors present in the mammalian brain.


Gradient expression of Cdx along the rat intestine throughout postnatal development.

Rat genomic DNA was isolated by homology with Cdx1, a murine homeogene selectively expressed in intestinal cells of endodermal origin. Southern blot analysis indicated that the rat genome contains a single or a small number of closely related Cdx gene(s). A major 1.7 kb Cdx mRNA was detected in neonate, suckling and adult rats whereas a 6.5 kb mRNA was restricted to sucklings and adults. Both transcripts showed decreasing concentration from the colon towards the proximal part of the small intestine. No obvious correlation could be established with the patterns of expression of transcripts corresponding to markers of cell proliferation and cell differentiation during postnatal development.


Intrastriatal dopamine-rich grafts induce a hyperexpression of Fos protein when challenged with amphetamine.

The aim of the present experiment was to characterize the effect of intrastriatal grafts of embryonic dopaminergic neurones on the expression of Fos protein in the striatum when challenged with amphetamine. Unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway were made in adult rats and grafting was performed 3 weeks later. The numbers of Fos-positive nuclei in the ipsi- and contralateral striata were counted on coronal sections following immunohistochemical staining 5 months after grafting. Administration of d-amphetamine induced an increase in the density of Fos-positive nuclei in the intact striatum. This stimulatory effect of amphetamine on c-fos expression was blocked by 6-hydroxydopamine hydrobromide lesions and was restored in the striata bearing transplants. However, an overshoot was observed as the density of Fos-positive cells within the grafted striatum was larger than that observed within the intact striatum. This hyperexpression of Fos-positive nuclei was correlated with the exaggerated compensation of amphetamine-induced rotation in the same animals.


Gamma-COP, a coat subunit of non-clathrin-coated vesicles with homology to Sec21p.

Constitutive secretory transport in eukaryotes is likely to be mediated by non-clathrin-coated vesicles, which have been isolated and characterized [(1989) Cell 58, 329-336; (1991) Nature 349, 215-220]. They contain a set of coat proteins (COPs) which are also likely to exist in a preformed cytosolic complex named coatomer [(1991) Nature 349, 248-250]. From peptide sequence and cDNA structure comparisons evidence is presented that one of the subunits of coatomer, gamma-COP, is a true constituent of non-clathrin-coated vesicles, and that gamma-COP is related to sec 21, a secretory mutant of the yeast Saccharomyces cervisiae.


[Regulation of growth hormone-releasing hormone gene expression and secretion].

Growth hormone (GH)-releasing hormone (GRH) is a stimulatory hypothalamic hypophysiotropic hormone which, along with an inhibitory peptide, somatostatin (SRIF), regulates the synthesis and secretion of GH in anterior pituitary somatotrophs. Although GHRH genes in several species have been characterized, there is only a limited understanding of the neural and hormonal mechanisms regulating GRH biosynthesis and secretion. Recent progress in PCR and in situ hybridization techniques as well as hGRF-transgenic animal models have provided an opportunity to study the regulation of GRH gene expression and secretion as well as its metabolism. The difference in 5'-untranslated sequences in both mouse and rat GRH cDNAs from hypothalamus and placenta has also suggested tissue-specific regulation of the GRH gene. GH excess has been shown to result in a decrease in hypothalamic GRH mRNA as well as GRH content and secretion while GH deficiency caused by hypophysectomy, hypothyroidism or genetic dwarfism causes an increase in GRH mRNA levels as tested by Northern blot analysis or in situ hybridization. Treatment of animals with GH or SRIF inhibits the increased GRH gene expression in the hypothalamic arcuate nucleus. Double immunocytochemistry for hypothalamic GRH and SRIF has shown both axo-perikaryal and axo-axonal connections between GRH- and SRIF- containing neurons. SRIF binding and GH receptor mRNA are demonstrated on a subpopulation of GRH-containing neurons in the hypothalamic arcuate nucleus. It is therefore possible to conclude that regulation of GRH gene expression, primarily related to inhibitory feedback effects of GH and IGFs on hypothalamic GRH gene expression, is mediated at least in part by SRIF or GH. The single transcript of the human GRH gene encodes a 108 amino acid precursor, prepro-hGRH, which is cleaved into the signal peptide and the remaining peptide, pro-hGRH. The latter is further processed to yield two equipotent forms of the releasing hormone, hGRH(1-44)-NH2, hGRH(1-40)-OH, and a carboxyl-terminal peptide (hGCTP) of unknown function. Studies in transgenic mice demonstrate the processing of hGRH-prohormone into both mature forms of hGRH and hGCTP, and provide evidence that hGRH(1-40)-OH is derived from hGRH(1-44)-NH2.(ABSTRACT TRUNCATED AT 400 WORDS)


Intradomestic mosquito breeding sources and their management.

Seven mosquito species were found to breed in intradomestic breeding sources. An. stephensi and An. subpictus bred in almost all types of containers. Among the culicines, Cx. quinquefasciatus and Ae. aegypti were predominant. Larval breeding was controlled through source reduction or introduction of larvivorous fish Poecilia reticulata. Health education helped in acceptance of the programme by the community. Intradomestic breeding positivity was 2-6% and 16-31% in experimental and control areas respectively.


Regulation of growth hormone secretion.

The secretion of growth hormone (GH) is controlled by a complex regulatory system. The primary control is exerted by two neuroendocrine hormones, GH-releasing hormone and somatostatin, though other hypothalamic neuropeptides directly and indirectly participate in this process. The regulation of each of these neurohormones is now being clarified at both physiologic and molecular levels, as are their respective roles in the generation of pulsatile GH secretion and in GH feedback regulation. Considerable information has been amassed concerning signal transduction systems mediating the effects of hypothalamic hormones on GH secretion. Although multiple second messengers have been implicated, the adenylate cyclase-cyclic AMP-protein kinase A system appears to exert a predominant role. The developmental regulation of the somatotropes and of GH gene expression is also of importance in determining the GH responses to releasing and inhibiting hormones. The availability of several rodent strains with genetic disorders of growth associated with impaired GH secretion, along with the development of transgenic models, has permitted a more detailed analysis of the role of cytokines and growth factors on both somatotrope biology and hormone secretion. Finally, knowledge gained from studies in animals is permitting a better understanding of the mechanisms involved in physiologic GH secretion and altered GH secretion associated with specific disease states in humans.


The role of prophylactic oophorectomy in the avoidance of ovarian cancer.

To determine the role of prophylactic oophorectomy at elective hysterectomy in the avoidance of ovarian cancer. A survey was performed of all members of the Society of Gynecologic Oncologists and all obstetrician-gynecologists in the State of Connecticut. Seventy-nine percent of gynecologic oncologists and 72% of obstetrician-gynecologists surveyed reported 0-1% of women would be saved from ovarian cancer if one ovary was removed at elective hysterectomy at age 45 years. If both ovaries were removed, 8.4% and 32.1% of these physicians respectively responded that 95-100% of women could be saved from ovarian cancer. Approximately 50% responded that macroscopic appearance and frozen section studies at surgery were helpful in deciding whether to remove ovaries at hysterectomy. A literature review suggested that 12% of ovarian cancers might be avoided if women undergo prophylactic oophorectomy at elective hysterectomy. There is a lack of well-designed epidemiologic studies evaluating risk factors for ovarian cancer and the role of prophylactic oophorectomy at elective hysterectomy.


Medication during pregnancy: an intercontinental cooperative study. Collaborative Group on Drug Use in Pregnancy (C.G.D.U.P.).

To assess the current situation in pregnancy drug prescription across various cultural and health care settings. An international study was set up to collect by questionnaire survey comparable data. A total of 14,778 women giving birth in 148 hospitals from 22 countries were enrolled. Antenatally, 14% of women received no drugs, while drug takers received an average of 2.9 prescriptions. There were marked intercountry variations in prescribing habits. The majority of prescriptions referred to iron and vitamins. Anti-infectives were the second most widely taken drugs. Anti-inflammatory drugs were taken by 17% of women, these in 42% of cases being self-administered. During the intrapartum period 79% of the women received an average of 3.3 drugs. Besides analgesics/anesthetics (31.8%), the most commonly prescribed drugs were oxytocin (17.5%), ergot derivatives (8.4%) and anti-infectives (5.3%). At the time of interview 91% of women were planning to breastfeed. Methylergometrin led the list of most frequently used drugs (36%) given to breastfeeding women, although the use of ergot derivatives in the puerperium showed wide intercountry variations. The survey has confirmed that at present, some drugs are often more widely used in pregnancy than is justified by the knowledge available.


Maternal mortality in Assiut.

Twenty-nine maternal deaths were identified among 8656 pregnant women residing in Assiut city and three surrounding villages (Upper Egypt). This gives a maternal mortality ratio of 368 per 100,000 live births. Of these maternal deaths 83% were due to direct obstetric causes (hemorrhage, eclampsia, ruptured uterus and sepsis). Logistic regression analysis showed that residence (in villages versus Assiut city), parity (nullipara and grandmultipara) and illiteracy were significantly associated with increased risk of maternal death.


The obstetric fistula: factors associated with improved pregnancy outcome after a successful repair.

A retrospective study of 155 pregnant patients with obstetric fistulae was conducted to determine the factors associated with improvement in the pregnancy outcome. The successful repair of the fistula was associated with a reduced abortion rate (4.0%), incidence of premature rupture of the membranes (1.3%) perinatal mortality (13.0%) as well as an increase in the mean fetal birthweight and reduced incidence of low birth (20.3%). Antenatal supervision of pregnancy increased the acceptance rate for elective cesarean section and was associated with early referral of cases to hospital during labor. The patients having their first pregnancies since the onset of their fistulae delayed longest in labor at home (15.6 h) hence had the highest perinatal mortality and rate of recurrence of obstetric fistula. Therefore the successful repair of the obstetric fistula together with an aggressive drive to improve antenatal supervision especially directed at the younger patients will improve acceptance of the policy of elective cesarean delivery and therefore the overall pregnancy outcome among these patients.


Racial differences in patients with adenocarcinoma of the endometrium.

To determine the differences between white and black women with regard to the presentation and behavior of adenocarcinoma of the endometrium. Records of 273 (68%) white patients and 117 (32%) black patients with endometrial adenocarcinoma were reviewed in Bloemfontein, South Africa. Survival data was calculated according to the direct method where losses in follow-up were regarded as tumor deaths. Most patients (82%) were treated by pre-operative radium followed by total abdominal hysterectomy and bilateral salpingo-oophorectomy, with post-operative external irradiation where indicated. Pre-operatively, fewer black women had reached FIGO stage I, while a larger number had advanced to stages II-IV (P = 0.0024). In addition, the tumor differentiation was more often poor in the black group (P < 0.0001). Ten-year follow-up was achieved in 84% of the white patients and 51% of the black patients and the 10-year survival figures were 67% for white patients and 28% for blacks (P < 0.0001). Endometrial adenocarcinoma is a more aggressive disease in black women than it is in whites.


Lipid and hormonal profile of Panamanian women during the menstrual cycle.

Forty-one women of reproductive age were included in the study, to establish the variation of serum lipids during the menstrual cycle and simultaneously, to determine the physiological fluctuation of FSH, LH, prolactin (PRL), progesterone (P4) and estradiol (E2) concentration in serum, during the menstrual cycle. A significant decrease of total serum cholesterol (165.29 +/- 3.6 mg/dl) and triglycerides (108.99 +/- 9.65) occurred during the luteal phase, as compared with the follicular phase (176.16 +/- 3.51, 108.99 +/- 9.65). Changes were not observed with HDL-cholesterol during the cycle. On the other hand, FSH showed an initial rise (mean 5 IU/l) followed by a surge (10 IU/l) and a progressive fall toward the midcycle. In contrast LH secretion showed a steady increase with a maximal concentration at surge (32.1 IU/l). PRL mean value was observed, with a discrete increase after day 13 of the menstrual cycle, that was more noticeable at the end of the cycle. Forty-eight hours after the FSH and LH midcycle surge, elevation of progesterone was observed, with maximal concentration occurring on day 24 (23 nmol/l) and later on progesterone levels fell rapidly. Thirty-six to 24 h before the surge of LH and FSH at midcycle was observed the peak serum concentration of estradiol (1300 pmol/l) followed by a progressive fall. Changes in the concentration of serum lipids during the menstrual cycle are presumably due to a direct or indirect effect of physiological fluctuation of sex hormones.


In vitro fertilization program based on programmed cycles monitored by ultrasound only.

Programmed oocyte retrieval was performed in order to make the in vitro fertilization (IVF) program cheaper and work of the IVF team easier. In a group of 77 patients included in the IVF program, the menstrual cycle was modified with estrogen-progesterone contraceptive pills. For this reason, it was possible to start the stimulation protocol in all patients on the same day. The stimulation protocol was a combination of clomiphene (100 mg) for 5 days and HMG (150 IU) every other day. Cycles were monitored by ultrasound only. The implantation rate per embryo transfer was 22.4%. The number of embryos per embryo transfer was low (2.6 +/- 1.4) and eliminated the need for cryopreservation. Fertilization rate (82%) and embryo transfer rate (87%) were high. The take home baby rate was 14.3%. Seventy percent of all punctures were performed in 3 days in the middle of the week. In our conditions, programmed oocyte retrieval is associated with significant economic benefits.


Carcinoma of the breast. ACOG Technical Bulletin Number 158--August 1991.

In the United States, breast cancer is the leading cause of cancer death in women aged 35-54. The cause is not known, and 80% of women with breast cancer do not have any of the known risk factors. Mass screening with mammography is the only effective method of reducing breast cancer mortality. Early detection discovers smaller lesions for which breast-sparing treatments may be appropriate. The obstetrician-gynecologist should recognize the breast examination is an integral part of the gynecologic examination.


Thyroid stimulating immunoglobulins, like thyrotropin activate both the cyclic AMP and the PIP2 cascades in CHO cells expressing the TSH receptor.

In human thyrocytes and in a permanent CHO cell line expressing the human thyroid stimulating hormone (TSH) receptor cDNA (JP09 cells), TSH activates both the cyclic AMP and the phosphatidylinositol 4,5-bisphosphate (PIP2) cascade, although the latter effect requires higher TSH concentrations. Thyroid stimulating autoantibodies (TSAb) activate also the human thyroid leading to the hyperthyroidism of Graves' disease. They bind to the TSH receptor and mimic the TSH stimulation of the gland by increasing intracellular cyclic AMP, but they do not enhance PIP2 hydrolysis in human thyroid slices. We show in this study that TSAb are able to activate the PIP2 cascade in JP09 cells, a cell line expressing high levels of TSH receptor. This suggests that the mechanism of action of TSAb on the TSH receptor is qualitatively similar to that of TSH.


Somatostatin binding and modulation of adenylate cyclase in ovine retina membranes.

Somatostatin (SS) receptors in membranes from ovine retinas were examined using 125I-Tyr11-SS as a ligand. Receptor binding was rapid, specific, saturable, reversible and dependent on temperature and membrane concentration. Conditions of apparent equilibrium were obtained at 25 degrees C after a 45 min incubation in the presence of about 0.25 mg membrane protein/ml. Native SS competitively inhibited the binding of 125I-Tyr11-SS in the range of 0.01-10 nM, and half-maximal inhibition was observed at 0.2 nM SS. Scatchard analysis of these data suggested the existence of a single population of SS receptors with a dissociation constant of 0.23 +/- 0.03 nM and a maximum binding capacity of 84 +/- 6 fmol/mg protein. The binding of 125I-Tyr11-SS was inhibited by various synthetic SS analogs in a dose-dependent manner whereas peptides unrelated to SS did not show practically any effect even at concentrations as high as 10(-6) M. SS receptor occupancy appears to be coupled to inhibition of adenylate cyclase activity by a guanine nucleotide-binding regulatory protein, as suggested by the facts that: (a) SS noncompetitively inhibited the stimulatory effect of vasoactive intestinal peptide (VIP) (3 x 10(-7) M) on membrane adenylate cyclase activity but it did not alter basal enzyme activity; and (b) the addition of guanosine 5'-triphosphate (GTP) (10(-5) M) decreased the specific binding of 125I-Tyr11-SS to 26.6% of the control value due to a decrease in SS receptor affinity. The present results support the hypothesis that SS may contribute to the physiological regulation of the functions of the retina.


Growth hormone and prolactin stimulate androgen receptor, insulin-like growth factor-I (IGF-I) and IGF-I receptor levels in the prostate of immature rats.

In this study we investigated the involvement of several different pituitary hormones on rat prostate development. 22-day-old Wistar rats, hypophysectomized (hypox) at 19 days of age were supplemented with highly purified human prolactin (hPRL), human luteinizing hormone (hLH), porcine follicle-stimulating hormone (pFSH), and bovine growth hormone (bGH) or with saline. Quantitative analysis of RNAs shows that treatment with either PRL or GH increases significantly steady-state mRNAs levels of the following genes in the prostate: androgen receptor (AR) (respectively 3.5- and 4.8-fold above hypox controls), IGF-I (5- and 2.7-fold), and IGF-I receptor (2.9- and 2.3-fold). LH and FSH, by contrast, have negative effects on these parameters. To test whether the enhancing effect of PRL and GH on AR-mRNA abundance was followed by increased content in the protein itself, binding assays were performed with the androgen agonist [3H]R1881 (131 and 153 fmol/mg protein while hypox controls contained 110 fmol/mg protein). In addition to the well-documented presence of prolactin receptors in prostatic tissues, we have further demonstrated, by means of nuclease S1 protection assays plus dot- and Northern-blot analyses, that a GH receptor mRNA is produced in the immature rat prostate. Moreover, we observed not only strong lactogenic but also purely somatogenic binding to be occurring in the immature prostates. Finally, we have studied IGF-I mRNA content in separated epithelial/stromal cell fractions and have concluded that IGF-I expression is principally located in the prostatic stroma. Taken together, these results suggest that PRL and GH are involved in regulating AR synthesis, at least partially by direct action on the organ. In this context IGF-I appears as a paracrine factor playing a role in epithelium/stroma interactions during prostatic development.


Identification and characterization of GroEL and DnaK homologues in Thiobacillus ferrooxidans.

The major heat shock proteins from Thiobacillus ferrooxidans were identified as DnaK and GroEL equivalents by Western blotting and analysis of the N-terminal amino acid sequence of spots isolated from dried 2-D polyacrylamide electrophoresis gels. The T. ferrooxidans chaperonins showed 70% and 80% identity with the Escherichia coli GroEL and DnaK, respectively. By using electrophoresis with a transverse pore gradient of cross-linked polyacrylamide and nondenaturing conditions followed by Western blotting, we found that the GroEL proteins from both bacteria formed a 14-mer, whereas E. coli DnaK protein existed partially as a dimer and the T. ferrooxidans DnaK-equivalent showed only a monomeric nature under our experimental conditions.


Generation of a restriction fragment length polymorphism linkage map for Toxoplasma gondii.

We have constructed a genetic linkage map for the parasitic protozoan, Toxoplasma gondii, using randomly selected low copy number DNA markers that define restriction fragment length polymorphisms (RFLPs). The inheritance patterns of 64 RFLP markers and two phenotypic markers were analyzed among 19 recombinant haploid progeny selected from two parallel genetic crosses between PLK and CEP strains. In these first successful interstrain crosses, these RFLP markers segregated into 11 distinct genetic linkage groups that showed close correlation with physical linkage groups previously defined by molecular karyotype. Separate linkage maps, constructed for each of the 11 chromosomes, indicated recombination frequencies range from approximately 100 to 300 kb per centimorgan. Preliminary linkage assignments were made for the loci regulating sinefungin resistance (snf-1) on chromosome IX and adenine arabinoside (ara-1) on chromosome V by linkage to RFLP markers. Despite random segregation of separate chromosomes, the majority of chromosomes failed to demonstrate internal recombination events and in 3/19 recombinant progeny no intramolecular recombination events were detected. The relatively low rate of intrachromosomal recombination predicts that tight linkage for unknown genes can be established with a relatively small set of markers. This genetic linkage map should prove useful in mapping genes that regulate drug resistance and other biological phenotypes in this important opportunistic pathogen.


Reduced levels of DNA polymorphism and fixed between-population differences in the centromeric region of Drosophila ananassae.

We have estimated DNA sequence variation within and between two populations of Drosophila ananassae, using six-cutter restriction site variation at vermilion (v) and furrowed (fw). These two gene regions are located close to the centromere on the left and right X chromosome arms, respectively. In the fw region, no DNA polymorphism was detected within each population. In the v region, average heterozygosity per nucleotide was very low in both populations (pi = 0.0005 in the Burma population, and 0.0009 in the India population). These estimates are significantly lower than those from loci in more distal gene regions. The distribution of DNA polymorphisms between both populations was also striking. At fw, three fixed differences between the Burma and India populations were detected (two restriction site differences and one insertion/deletion of approximately 2 kb). At v, each DNA polymorphism in high frequency in the total sample was nearly fixed in one or the other population, although none of them reached complete fixation. The observed pattern of reduced variation within populations and fixed differences between populations appears to correlate with recombination rate. We conclude that recent hitchhiking associated with directional selection is the best explanation for this pattern. The data indicate that different selective sweeps have occurred in the two populations. The possible role of genetic hitchhiking in rapid population differentiation in gene regions of restricted recombination is discussed.


Comparative genome mapping of Sorghum and maize.

Linkage relationships were determined among 85 maize low copy number nuclear DNA probes and seven isozyme loci in an F2 population derived from a cross of Sorghum bicolor ssp. bicolor x S. bicolor ssp. arundinaceum. Thirteen linkage groups were defined, three more than the 10 chromosomes of sorghum. Use of maize DNA probes to produce the sorghum linkage map allowed us to make several inferences concerning processes involved in the evolutionary divergence of the maize and sorghum genomes. The results show that many linkage groups are conserved between these two genomes and that the amount of recombination in these conserved linkage groups is roughly equivalent in maize and sorghum. Estimates of the proportions of duplicated loci suggest that a larger proportion of the loci are duplicated in the maize genome than in the sorghum genome. This result concurs with a prior estimate that the nuclear DNA content of maize is three to four times greater than that of sorghum. The pattern of conserved linkages between maize and sorghum is such that most sorghum linkage groups are composed of loci that map to two maize chromosomes. This pattern is consistent with the hypothesized ancient polyploid origin of maize and sorghum. There are nine cases in which locus order within shared linkage groups is inverted in sorghum relative to maize. These may have arisen from either inversions or intrachromosomal translocations. We found no evidence for large interchromosomal translocations. Overall, the data suggest that the primary processes involved in divergence of the maize and sorghum genomes were duplications (either by polyploidy or segmental duplication) and inversions or intrachromosomal translocations.


High density molecular linkage maps of the tomato and potato genomes.

High density molecular linkage maps, comprised of more than 1000 markers with an average spacing between markers of approximately 1.2 cM (ca. 900 kb), have been constructed for the tomato and potato genomes. As the two maps are based on a common set of probes, it was possible to determine, with a high degree of precision, the breakpoints corresponding to 5 chromosomal inversions that differentiate the tomato and potato genomes. All of the inversions appear to have resulted from single breakpoints at or near the centromeres of the affected chromosomes, the result being the inversion of entire chromosome arms. While the crossing over rate among chromosomes appears to be uniformly distributed with respect to chromosome size, there is tremendous heterogeneity of crossing over within chromosomes. Regions of the map corresponding to centromeres and centromeric heterochromatin, and in some instances telomeres, experience up to 10-fold less recombination than other areas of the genome. Overall, 28% of the mapped loci reside in areas of putatively suppressed recombination. This includes loci corresponding to both random, single copy genomic clones and transcribed genes (detected with cDNA probes). The extreme heterogeneity of crossing over within chromosomes has both practical and evolutionary implications. Currently tomato and potato are among the most thoroughly mapped eukaryotic species and the availability of high density molecular linkage maps should facilitate chromosome walking, quantitative trait mapping, marker-assisted breeding and evolutionary studies in these two important and well studied crop species.


A retrospective comparative study of reconstructive methods following pancreaticoduodenectomy--pancreaticojejunostomy vs. pancreaticogastrostomy.

Reconstructive methods following pancreaticoduodenectomy in our department are discussed and evaluated in this study. Between January 1980 and November 1990 fifty-two consecutive patients underwent pancreaticoduodenectomy because of pancreas head disease. Thirty-one patients underwent pancreaticojejunostomy and twenty-one had pancreaticogastrostomy as reconstructive procedures. Mortality rate was 6% in pancreaticojejunstomy versus zero in pancreaticogastrostomy. Six patients had leakage from the pancreaticojejunostomy, but only one patient had necrosis of the gastric stump and leakage from the pancreaticogastrostomy. This case had previous distal gastrectomy done for gastric ulcer. The residual stomach might not have been large enough, and the blood supply of the gastric stump might not have been adequate for pancreaticogastrostomy. Except for this case, none was observed with leakage from the pancreatic anastomosis in the pancreaticogastrostomy group. No statistical significance in operating time or blood loss was observed between the two methods. The pancreaticogastrostomy cases without complications had significantly less loss of body weight than those with pancreaticojejunostomy at the date of discharge (p < 0.05). It is concluded that pancreaticogastrostomy is the safer reconstructive method following pancreaticoduodenectomy, although it may not be indicated in patients with prior gastrectomy.


Conservative treatment of acute pancreatitis: the use of somatostatin.

The aim of the medical treatment of acute pancreatitis is to rest the gland and to reduce complication and mortality rates. The effect of various treatment modalities on the course and outcome of acute pancreatitis have been disappointing. This review critically considers the results of reported therapeutic trials and our own unpublished data of somatostatin and its long-acting synthetic analogue, octreotide, in acute pancreatitis. It is concluded that somatostatin is a promising drug. It reduces pancreatic enzyme secretion, the clinical need for analgesic drug administration, and the local complication rate, and shortens hospitalization. However, its effect on mortality rates is questionable. It is suggested that large-scale, carefully designed studies of somatostatin are needed to evaluate the beneficial effect of this drug on the course and outcome of acute pancreatitis. Since most of the trials included pancreatitis of various etiologies, it is also suggested that cases of acute biliary pancreatitis should be excluded or evaluated separately, as these patients are best treated by endoscopic sphincterotomy.


Psychotic depression: advances in conceptualization and treatment.

Psychotic depression is a unique subtype of depressive illness in which mood disturbance is accompanied by delusions, hallucinations, or both. Once considered relatively uncommon, it is frequently encountered in clinical practice, particularly in treatment-resistant depressed patients. Psychotically depressed patients respond poorly to antidepressants, but remission is likely with neuroleptic-antidepressant combinations or electroconvulsive therapy. Psychotic depression may be unipolar or bipolar with early or late onset and may be more likely to occur in patients with a history of childhood psychic trauma. Much is known about the course and treatment response of obvious presentations of psychotic depression, but more must be learned about depressed patients who have intermittent, subtle, or mild psychotic symptoms and about the ways in which the capacity to become psychotic interacts with the capacity to become depressed to produce a syndrome greater than the sum of its parts.


Differences between chronic schizophrenic patients in the hospital and in the community.

Clinical differences between stable, chronic schizophrenic patients with long stays in the hospital and schizophrenic patients living in the community were investigated. Patients were matched for age, gender, and diagnosis. Hospitalized patients had more severe thought disorder and negative symptoms, and those in the community had a significantly higher incidence of depression and anxiety. The community-based patients were also receiving higher doses of neuroleptic drugs and had a higher incidence and severity of extra-pyramidal side effects. Results suggest that living in the community, despite its obvious benefits, may have its price in terms of the distressing effects of affective symptoms and neuroleptic side effects.


Linkage analysis of the D1 dopamine receptor gene and manic depression in six families.

Disturbances in dopaminergic activity may play an important role in the pathogenesis of manic depression. The effects of dopamine are mediated by at least five G protein coupled receptors, D1, D2, D3, D4 and D5. Recently, three separate research groups have cloned and characterized the D1 dopamine receptor, which localizes to 5q35.1. We undertook a linkage analysis between the D1 receptor polymorphisms and manic depression in six families in which segregation of the disease was consistent with autosomal dominant inheritance. A highly polymorphic flanking DNA marker, CRI-L1200, was also analyzed as the D1 gene RFLPs were relatively uninformative in our families. Multipoint analyses of manic depression and these DNA markers resulted in lod scores of less than -3.0 at the D1 locus, indicating that the D1 dopamine receptor gene does not confer an inherited susceptibility to manic-depressive illness in the families studied.


Leber's hereditary optic neuroretinopathy and the X-chromosomal susceptibility factor: no linkage to DXs7.

Leber's hereditary optic neuroretinopathy (LHON) was the first human disease for which mitochondrial inheritance was demonstrated. Analysis of genealogies, however, suggests the existence of an interacting X-linked factor, and linkage to DXS7 was recently described. We tested this location in four LHON families, with DXS7 and two flanking markers, OTC and DXS426. We found recombinations with DXS7 in two families and with DXS426 in one. The two point lod scores to DXS7 were negative with all the allele frequencies for the X-linked factor tested (q = 0.5; 0.35; 0.05).


Haemopoietic stem cells during development of mouse embryo.

Haemopoiesis in mammals takes place in yolk-sac and in mouse it can be detected on the 7th day of gestation. Erythropoietin (EPO) responsive cells can be detected from 7th day onwards. However, the cells committed to the myeloid lineage which can respond to the haemopoietic growth factor (viz. granulocyte macrophage colony stimulating factor; GM-CSF) can be demonstrated only on 10th day of gestation. At the same time, the 12-day spleen colony forming cells i.e. the late colony forming unit spleen (CFU-s) which are multipotent stem cells can also be detected. Data suggest that the stem cells seen in the embryo from 7-10 days of gestation may be a primitive population confined only to the yolk-sac. Liver haemopoiesis which begins in the liver of 13-day embryos is due to primitive haemopoietic pluripotent stem cells, arising de novo in the embryo and not in the yolk-sac, since no primitive pluripotent stem cells capable of repopulating lethally irradiated bone-marrow can be detected in the yolk-sac.


Dopaminergic modulation of footshock induced aggression in paired rats.

Footshock induced aggression (FIA) was induced in weight matched paired rats and three paradigms of aggressive behaviour was recorded, namely, the latency to fight (LF), total period of physical contact (TPP) and cumulative aggression scores (CAS). Dopamine (DA), administered centrally, and peripherally administered L-dopa (with benserazide, a peripheral decarboxylase inhibitor), a DA precursor, and the postsynaptic D2 receptor agonists, apomorphine, N-n-propyl-norapomorphine (PNA), bromocriptine, lisuride and pergolide, induced a dose-related facilitation of FIA characterized by decrease in LF and increase in TPP and CAS. However, the DA presynaptic receptor agonist, BHT-920, induced a biphasic effect with inhibition of FIA being induced by a lower dose and facilitation of the aggressive behaviour produced by a higher dose. The postsynaptic D2 receptor antagonists, haloperidol, spiperone and pimozide, induced a dose-related attenuation of FIA, an effect not seen with domperidone, a peripheral DA receptor antagonist. The results indicate that central dopaminergic postsynaptic D2 receptors have a modulatory facilitative effect on FIA, while the presynaptic DA autoreceptors mitigate aggressive behaviour. However, the presynaptic DA receptor agonist, BHT-920, appears to lose its receptor specificity on dose increment. Long term administration of haloperidol, followed by withdrawal, or desipramine, induced per se augmentation of FIA and potentiated the aggression-facilitative effects of L-dopa, apomorphine and PNA. Since both these treatments are known to induce supersensitivity of central postsynaptic dopamine D2 receptors, the effects are likely to be related to augmented function of dopamine neurones. The findings, in conjunction with a recent report from this laboratory indicating an increase in rat brain DA levels in FIA, support the contention that the central DA system has a facilitative effect on FIA.


Central histaminergic involvement during stress in rats.

Effects of some drugs modulating central histaminergic (HA) transmission were evaluated on restraint stress (RS)-induced gastric ulcerogenesis, plasma corticosterone and immune responses in rats. RS for (i) 6 hr or (ii) 24 hr at room temperature, and (iii) 3 hr at 4 degrees C (CRS) all induced gastric mucosal erosions and elevated plasma corticosterone levels, the effects with the latter two RS procedures being most consistent. Pretreatment of rats with neuronal HA depletor, alpha-FMH (100 mg/kg, ip) attenuated both ulcer severity and corticosterone response, during both 24 hr RS and CRS. Similar effects were also seen with the mast cell degranulator, C-48/80 (10 micrograms/kg, i.c.v.) treatment. Further, the H1-blocker, pheniramine (25 mg/kg, ip) but not the centrally acting H2-blocker, zolantidine (5 mg/kg, ip) produced clearcut attenuations in both stress markers, during the experimental stressors. In rats immunized in SRBC, 24 hr RS (and not CRS) significantly prevented the humoral immune responses to the antigen. alpha-FMH, C 48/80 and pheniramine but not zolantidine, reversed this response during 24 hr RS. The results indicate a central HA ergic involvement in the visceral, endocrinal and immune responses during RS and suggest the probable role of both neuronal as well as extraneuronal (mast cell) HA and activation of H1-receptors in the mediation of these effects.


Testosterone secretion in children with undescended testis.

Testicular testosterone (T) production was examined in thirty boys with undescended testes (UT) following the administration of 4500 U gonadotropic hormone. Twenty boys had bilateral UT and ten had UT plus hypospadias. As for possible causes of reduced Leydig cell activity it was investigated whether the testis was (1) hypoplastic; (2) abnormally fused with the epididymis; (3) located in the abdomen; (4) or UT was associated with hypospadias. Average T values were significantly lower when the testicle was hypoplastic or its fusion with the epididymis was imperfect; but remained largely undiminished when the testicle was located in the abdomen or when UT was combined with hypospadias. The occurrence of both pathologic and physiologic T reactions in each of the four groups suggests that the population of UT children is heterogeneous, probably due to differences in aetiology and in intrauterine hormonal processes. In the case of UT and hypoplasia the time and method of operation (orchidopexy) must be selected with utmost care, bearing in mind that an originally small testicle with impaired T secretion may become physiologic by the time of puberty.


Detection of P-glycoprotein on endothelial and endocrine cells of the human pancreatic islets by C 494 monoclonal antibody.

P-glycoprotein, an integral membrane protein acting as an energy-dependent efflux pump, has been detected immunocytochemically in the human pancreatic islets using C 494 monoclonal antibody. Intense P-glycoprotein immunoreactivity was found in both endothelial cells of islet blood capillaries and in endocrine cells. Strong expression of P-glycoprotein has been found in the capillary blood vessels at blood-tissue barrier sites and in numerous kinds of cells with secretory/excretory function. Therefore the present findings suggest that P-glycoprotein may play a role in controlling the composition of the extracellular fluids and the intracellular milieu of endocrine islet cells and possibly in regulating their secretory activity.


Statistical associations between restriction fragment length polymorphisms and quantitative traits in beef cattle.

Data on 41 traits from 677 animals produced in a five-breed diallel were matched with genotypes for five marker-loci provided by restriction fragment length polymorphisms to detect quantitative effects associated with the markers, following three different designs based on inbred lines, half-sib families, and on assumptions of the markers being quantitative trait loci (QTL). Three growth hormone-TaqI alleles, B, C, and D, in high frequencies in this sample of the Brahman breed, were associated with decreases in birth weight, as a maternal trait (P < .01), and decreases in shoulder width at birth (P < .05). Among F2 Angus-Brahman and Brahman-Hereford cows, homozygotes for the B, C, or D alleles gave birth to calves 4.0 kg lighter than cows homozygous for the A allele, an effect that amounts to the magnitude of the corresponding breed difference in the diallel, and represents one phenotypic SD. A putative cytoplasmic effect seems to interact (P = .02) with this effect on maternal birth weight. Also, at birth, F2 calves homozygous for the B, C, or D alleles were .8 cm narrower at the shoulders than those homozygous for the A allele, after adjusting for birth weight. Significant associations (P < .05) between the parathyroid hormone-MspI marker and measures of body size were detected, as well as an effect on weaning weight (P = .03) as a maternal trait, whose magnitude (17.5 kg) equals the Brahman vs Angus and Hereford breed difference, as quantified in the diallel, and represents .8 of a phenotypic SD. No significant associations were found for three other marker-loci (prolactin-MspI, osteonectin-EcoRI, and keratin VI-MspI). Restriction fragment length polymorphisms have the potential to provide new insights and useful applications to animal breeding, but, as in this work, small sample sizes, extreme susceptibility to Type I errors, and different types of possible confounding obfuscate the conclusions that can be drawn from studies of limited scope and less than ideal planning.


Influence of genetic capacity for lean tissue growth on rate and efficiency of tissue accretion in pigs fed ractopamine.

The influence of genetic capacity for lean tissue (LT) growth on responses of pigs to ractopamine, in terms of rate and efficiency of body growth and the distribution and accretion rate of body tissues, was determined in this study. Two sources of pigs representing low and high LT genotypes were used. Within each source, two littermate barrows from each of eight litters were individually penned and given ad libitum access to a lysine-supplemented, corn-soybean meal diet (17.7% CP, 1.08% lysine) containing 0 or 20 ppm of ractopamine hydrochloride from 63 to 104 kg. Carcasses were physically dissected into muscle, fatty tissue, skin, and bone. Within each source, four additional pigs were killed for determination of initial body composition. Pigs of high LT genotype gained BW and muscle faster (P < .01), required less (P < .01) feed per unit of gain, and produced carcasses that contained more (P < .01) muscle and bone and less (P < .01) fatty tissue. Ractopamine increased (P < .01) weight gain and improved (P < .01) feed:gain ratio in both genotypes. Ractopamine enhanced the accretion rate and the amount of carcass muscle in both genotypes, but the degree of improvement was greater in pigs of the high than in those of the low LT genotype (genotype x ractopamine, P < .02). Ractopamine also reduced the accretion rate and amount of dissectible fat by a greater magnitude in the high LT genotype (genotype x ractopamine, P < .04). Based on these data, ractopamine increases muscle accretion to a greater degree in pigs with a high genetic capacity for LT growth than in those with a low capacity.