Dopamine stimulates growth hormone release from the pituitary of goldfish, Carassius auratus, through the dopamine D1 receptors.

Previously, we have demonstrated that ip injection of apomorphine, a nonselective dopamine (DA) agonist, increases serum GH levels in the goldfish, suggesting a possible role of DA in GH regulation. In the present study, the effects of DA on GH release in the goldfish were further characterized using an in vitro perifusion system for pituitary fragments. DA increased GH release in a dose-dependent manner with an ED50 of 0.26 +/- 0.06 microM. SKF38393, a DA D1 agonist, mimicked the GH-releasing effect of DA with an ED50 of 0.41 +/- 0.12 microM. Stereoselectivity consistent with mammalian DA D1 systems was demonstrated for the GH response to SKF38393; only the (+)- but not (-)-enantiomer of SKF38393 induced a dose-dependent GH release. Two other D1 agonists, SKF77434 and SKF82958, were also found to have GH-releasing activity. In contrast, high doses (up to 1 microM) of the DA D2 agonists, bromocriptine and LY171555, did not affect basal GH levels. The receptor specificity for DA-stimulated GH release was further investigated by using D1 and D2 antagonists; the D1 antagonists SCH23390 and SKF83566 completely abolished the GH response to DA or the D1 agonist SKF38393, whereas the D2-specific antagonists domperidone and (-)-sulpiride were not effective in this respect. Taken together, the present study demonstrates that DA is stimulatory to GH release from the pituitary of goldfish, and its action is mediated through receptors resembling the mammalian DA D1 receptors. The apparent similarities of the DA D1 receptor pharmacology between the goldfish and the mammals also indicate that D1 receptor is highly conserved during vertebrate evolution.


Metabolism of the anticoagulant peptide, MDL 28,050, in rats.

Rats were each administered a 9 mg/kg iv bolus dose of a 3H-labeled decapeptide anticoagulant, MDL 28,050. Tritium was eliminated rapidly with approximately 50% of the dose recovered in urine within the first 6 hr. Renal excretion accounted for 68% of the dose, whereas fecal excretion accounted for 16% of the dose. Continuous flow fast atom bombardment mass spectrometry was used to identify the major urinary metabolites of MDL 28,050. Trace amounts of parent drug were found, and other biotransformation products indicated that hydrolysis had occurred at four peptide bonds. Two initial sites of hydrolysis were identified as 4I-5P and 6E-7E, which resulted in the peptide fragments Suc-Y-E-P-I-OH + P-E-E-A-Cha-E-OH and Suc-Y-E-P-I-P-E-OH + E-A-Cha-E-OH, respectively. Further metabolism of these fragments resulted in the N-terminal pentapeptide and the C-terminal dipeptide.


Pyridostigmine-mediated growth hormone release: evidence for somatostatin involvement.

Pyridostigmine (PD), a cholinesterase inhibitor, has been shown to elicit GH release when given alone and to potentiate the GH response to GH-releasing hormone (GHRH) in man. Numerous experiments have indirectly indicated that somatostatin (SS) inhibition is its likely mechanism of action. This study sought to establish the ability of PD to induce GH release in the rat, determine the dose-response relationship, and test the hypothesis that SS inhibition is the method of action. Three experiments were performed to monitor the GH response to PD. I) Five groups of male rats were food deprived for 72 h. The groups were then treated iv with saline, SS antibody (SS-ab), and 10, 100, and 1000 micrograms/kg PD, respectively. Blood samples were drawn before and after treatment. II) Two groups of male rats were pretreated iv with GHRH antibody (GHRH-ab) and either SS-ab or normal sheep serum (NSS). Blood samples were drawn every 30 min for 8.5 h, during which time each animal was injected with PD (10 micrograms/kg) in the third hour and again in the sixth hour. III) Male rats received a PD injection (10 micrograms/kg, iv) during a spontaneous GH trough period and a second PD injection during a spontaneous GH peak period. Blood samples were drawn at regular intervals preceding and following treatments. In Exp I, PD induced a clear 4- to 5-fold increase in GH concentrations in food-deprived rats. The maximal GH responses occurred after the 10 and 100 micrograms/kg doses, although the pattern and duration were different with these two doses. In Exp II, PD induced an approximately 2-fold increase in GH values in animals pretreated with GHRH-ab and NSS, but failed to induce a change in GH in the animals treated with GHRH-ab and SS-ab. In Exp III, PD failed to induce any change in GH concentration when administered during spontaneous GH peaks or troughs. The first two experiments suggest that PD increases GH secretion in the rat via inhibition of SS. The failure of PD to alter GH during a spontaneous peak is consistent with the current hypothesis that the level of SS is low at this time. Its failure to alter GH during trough periods may be related to very high SS tone. In conclusion, our results support the hypothesis that PD acts via inhibition of SS secretion.


The urinary metabolic profile of metyrapone in the rat. Identification of two novel isomeric metyrapol N-oxide metabolites.

This study was designed to fully characterize the urinary metabolic profile of metyrapone following a 50 mg/kg ip dose to male Sprague-Dawley rats. Preliminary examination of alkaline dichloromethane extracts of urine by TLC and HPLC showed the absence of the intact drug, but the presence of moderate amounts of the two isomeric metyrapone mono-N-oxides, together with small amounts of metyrapol and the alpha-pyridone metabolite. These compounds have previously been reported as in vitro metabolites. Large amounts of two new metabolites of metyrapone were also observed and were conclusively identified as the isomeric metyrapol N-oxides by a combination of low- and high-resolution mass spectrometry. Quantitation by HPLC showed that metyrapol and the two metyrapone mono-N-oxides accounted for 4 and 10% of the dose, respectively. The two metyrapol mono-N-oxides accounted for more than 75% of the dose. These are novel metabolites not previously reported either as in vivo or in vitro metabolites.


Effects of ovarian steroids on the gonadotropin response to N-methyl-D-aspartate and on hypothalamic excitatory amino acid levels during sexual maturation in female rats.

In order to evaluate the involvement of estrogen-progesterone (EP) in the effects of N-methyl-D-aspartate (NMDA) receptor stimulation on gonadotropin secretion during sexual development in female rats, NMDA (30 mg/kg sc) was administered to 16- and 30-day-old female rats pretreated with EP. NMDA administration induced increases in plasma LH concentration that were 13.6-fold and 94.5-fold higher, respectively, than those found after NMDA alone. The increase of LH levels induced by NMDA was accompanied by a significant enhancement of the content of GnRH in the anterior and preoptic hypothalamic areas and in the medial basal hypothalamus (APOA/MBH). EP potentiated this increase of GnRH induced by NMDA. NMDA increased plasma FSH levels at 16 days of age, and this increase was inhibited by EP treatment. In 30-day-old rats EP induced FSH release in response to NMDA. This release was not observed in rats treated only with NMDA. In 16-day-old rats EP induced an increase in the concentrations of aspartate, glutamate, and glycine in the anterior and preoptic hypothalamic areas and in the medial basal hypothalamus, the excitatory amino acids involved in NMDA neurotransmission. This effect was not observed in rats of 30 days of age. In summary, the present results show that during sexual maturation ovarian steroids potentiated the LH-releasing response to NMDA probably by acting at the hypothalamic level; furthermore, during sexual maturation there are changes in the response to EP of the hypothalamic concentrations of excitatory amino acids. These findings could be related to the neuroendocrine mechanisms regulating the onset of puberty and the sexual cycle in female rats.


Felbamate metabolism in pediatric and adult beagle dogs.

In a metabolism study, three male and three female pediatric dogs and three male and three female adult beagle dogs received a single oral dose of 60 mg/kg [14C]felbamate. Urine and feces were collected up to 72 hr. Excreta samples were extracted with ethyl acetate and 14C metabolite profiles were generated by HPLC analysis of the extracts. The total 14C dose recoveries were 88.2% for the pediatric and 85.9% for the adult groups; recovery in urine accounted for 61.7 and 69.7%, respectively. The 14C metabolite profiles in urine and feces were similar, with three major peaks corresponding to unchanged felbamate and two hydroxylated metabolites. There was no significant sex difference in profiles within each age group. However, the sum amount of all metabolites in the urine was higher, and the amount of unchanged drug was lower in pediatric dogs. For the 0-24 hr samples, the metabolites/drug ratio in urine was 2.0-2.1 for pediatric dogs and 1.0-1.2 for adult dogs. This indicated a higher metabolic rate in the pediatric dogs.


Identification of cytochrome P-450IIB1 as a cocaine-bioactivating isoform in rat hepatic microsomes and in cultured rat hepatocytes.

Induction of cytochrome P-450-dependent monooxygenases with phenobarbital (PB) or other hepatic drug-metabolizing enzyme inducers in the rat is associated with enhanced cocaine hepatotoxicity both in vivo and in cultured rat hepatocytes. To demonstrate whether the major PB-inducible P-450 subfamily (P-450IIB) could be involved in the metabolic activation of cocaine, rates of cocaine N-demethylation (the first step of cocaine bioactivation) and the rate of irreversible (covalent) binding of tritiated cocaine to hepatic microsomal proteins (a measure for the overall bioactivation) were determined in microsomes from saline or PB-pretreated rats. PB pretreatment augmented Vmax (6-fold), but not KM, of cocaine N-demethylation. Similarly, the rate of irreversible protein binding was 3-fold increased in microsomes from PB-pretreated rats as compared with those from saline controls. Addition of benzphetamine, a substrate of P-450IIB, markedly inhibited cocaine irreversible binding. In addition, various concentrations of cocaine inhibited microsomal pentoxyresorufin O-depentylase activity in a competitive-type pattern. A polyclonal antibody raised against purified rat P-450IIB1 markedly inhibited cocaine N-demethylation as compared with control incubations with preimmune IgG. Finally, pretreatment of rats with PB potentiated cocaine-induced cytotoxicity in primary, short-term cultured hepatocytes, assessed as lactate dehydrogenase release into the culture medium. This enhancing effect of PB became even more evident in glutathione-depleted cells. These results suggest that cocaine is metabolized and bioactivated by P-450IIB1 in the rat liver, and that induction of this isoform with various agents may be associated with enhanced lethal hepatocyte injury in the rat.


Ontogeny of gastrin, somatostatin, and the H+/K(+)-ATPase in the ovine fetus.

In the term human and ovine fetus, plasma gastrin is elevated, but gastric acid secretion is below adult levels, suggesting a developmentally related immaturity in gastrin and gastric acid regulation. This study investigated a number of elements of the gastric acid regulatory system: gastrin and its glycine-extended precursor, somatostatin, and the H+/K(+)-ATPase. Measurements were made in blood, antrum, and fundus of the ovine fetus during the last half of gestation, of 15-day-old lambs, and of adult sheep at the level of mRNA synthesis, tissue storage, and secretion. Plasma amidated gastrin (gastrin-amide) was elevated at or above adult values from 125 days (term is 145 days) and steadily increased with development, peaking in the lamb. Similar changes occurred with plasma glycine-extended gastrin (gastrin-gly). The peak concentration of antral gastrin-amide was present in the lamb, while the maximum antral gastrin-gly level occurred 1 week before birth. Gastrin mRNA paralleled the changes in antral gastrin-gly. The proportion of higher mol wt species of gastrin decreased during gestation in both plasma and antrum. Low amounts of mRNA for the H+/K(+)-ATPase was present from at least 120 days of gestation and antedated gastric acid secretion. However, there was a 3-fold increase in H+/K(+)-ATPase mRNA from the 140-day-old fetus to the lamb, the period when the greatest reduction in gastric pH occurred (pH 5 to 2). Antral and fundic somatostatin increased rapidly in the fetus at 120 days gestation and were above adult values at term and in the lamb. Somatostatin mRNA changed in parallel to somatostatin peptide. Somatostatin-14 was the major species in antrum and fundus throughout development. The increase in circulating and antral gastrin-amide after birth may be the result of increased amidation of gastrin-gly as well as increased expression of gastrin mRNA. Amidation of gastrin may be a regulatory step in the production of biologically active gastrin during development. The major increase in gastrin and the H+/K(+)-ATPase that occurs in the week before and after gestation correlated with the onset of increased gastric acidity.


Seeding of neonatal lymph nodes by T cells and identification of a novel population of CD3-CD4+ cells.

Mature T cells first appear in the thymus of the mouse a few days before birth, about 7-8 days after the thymus was colonized by stem cells. These mature cells are exported to the peripheral lymphoid organs beginning at about the time of birth, but because the number is very small at this stage, little is known about the phenotype or function of these early emigrants. We have examined the cells that accumulate in the peripheral lymph nodes (LN) during the first week of life to understand better the initial seeding of the periphery by T cells. Our studies showed that a high proportion of neonatal LN cells were CD4+, but that the majority of these were CD3- during the first few days of life. The CD3- population did not increase greatly in number after birth and rapidly diminished in proportion as the number of CD3+ cells increased. These CD3-CD4+CD8- cells were found to be Thy-1loCD44+ and to lack surface expression of heat-stable antigen. B220 and Mac-1. They had lymphoid morphology, did not phagocytose latex, and did not exhibit any precursor activity for cells of hemopoietic lineages. Their origin (intra- or extrathymic) as well as their function and physiological role, therefore, remains unknown. CD3+ T cells, both CD4+CD8- and CD4-CD8+, were present in low numbers during the first 1-2 days of life, but at post-natal day 3, a sharp increase in the accumulation of these cells occurred in both LN and spleen. By day 3 the CD4:CD8 ratio in LN was about 2:1, as in the adult. Crude estimates of the rate of export from the thymus from day 3 onwards gave values around 1% of thymocytes per day, i.e. close to our previous estimates for young adult thymus. We found no evidence of particularly high levels of emigration from the thymus during the first week after birth. Both CD4+CD8- and CD4-CD8+ T cell subsets were present in the LN as early as 1 day post-natally with CD4-CD8+ predominating among LN T cells, even though CD3+CD4+CD8- cells predominated over CD3+CD4-CD8+ cells in the thymus. By day 3 the ratio had changed to 2:1 (as in the adult). T cells, therefore, appear to emigrate from the thymus from about the time of birth with a dramatic increase around day 3 after birth.


Altered course of visceral leishmaniasis in mice expressing transgenic I-E molecules.

Previous studies had shown that the outcome of infection with Leishmania donovani was exquisitely sensitive to the influence of the major histocompatibility complex. In this study, we have examined the course of infection in non-obese diabetic (NOD) and NOD-E-3 mice, the latter expressing an I-E molecule as a result of transgenic introduction of the wild-type Ed alpha gene. Introduction of this transgene significantly altered the course of infection allowing for enhanced parasite multiplication in the viscera from day 14 to day 28. This was associated with both a delayed and reduced tissue granulomatous response in NOD-E-3 mice. In vitro, spleen cells from these mice produced equivalent levels of interferon (IFN)-gamma during the early phase of infection but this originated from populations having a different balance of T cells subsets. In NOD mice CD8+ T cells contribute substantially to the total levels of IFN-gamma produced, but in transgenic mice the contribution from this subset is significantly decreased. This is reflected in a reduction in the proportion of Leishmania-specific CD8+ T cells, which could only partially be accounted for by deletion of V beta 5- and V beta 3-expressing CD8+ T cells in NOD-E-3 mice. This study highlights the impact of the introduction of a class II gene product on disease outcome and unexpectedly on the functional potential of CD8+ T cells.


Engagement of major histocompatibility complex class I and class II molecules up-regulates intercellular adhesion of human B cells via a CD11/CD18-independent mechanism.

We have studied the role of major histocompatibility complex (MHC) molecules in the regulation of intercellular adhesion of human B cells. We found that molecules able to bind to MHC class II molecules, such as monoclonal antibodies or staphylococcal enterotoxins, induced rapid and sustained homotypic adhesion of Epstein-Barr virus (EBV)-transformed B cell lines as well as peripheral blood B lymphocytes. Moreover, anti-MHC class I monoclonal antibodies also stimulated intercellular adherence. Adhesion induced upon MHC engagement was faster and stronger than that triggered by phorbol esters. It needed active metabolism, but divalent cations were not required. Monoclonal antibodies directed against LFA-1 (CD11a/CD18) or its ligand ICAM-1 (CD54) did not inhibit MHC class II-induced homotypic adhesion of various EBV-transformed B cell lines, nor of a variant of the B cell line Raji expressing very low LFA-1 surface levels. Moreover, EBV-transformed B cells from a severe lymphocyte adhesion deficiency patient, lacking surface CD11/CD18, also aggregated in response to anti-MHC class I or class II monoclonal antibodies. Together these data indicate that engagement of MHC molecules may transduce signals to B cells resulting in up-regulation of intercellular adhesion, via an LFA-1-independent mechanism. This may play a role in the stabilization of T cell/antigen-presenting cell conjugates at the moment of antigen recognition.


A unique CD44 monoclonal antibody identifies a new T cell activation pathway.

We have identified a new T cell activation pathway mediated by the lymphocyte homing receptor/CD44 molecule, 8B2.5, a local monoclonal antibody (mAb), which recognizes two glycoproteins of 85 and 220 kDa with wide tissue distribution, is shown by sequential immunoprecipitations and competitive antibody-binding inhibition experiments with several CD44 reference mAb to recognize the CD44 molecule. The 8B2.5 mAb, but not reference CD44 mAb, is able to induce resting peripheral blood lymphocytes to proliferate in the presence of phorbol esters. This proliferation is monocyte dependent but Fc independent and results from 8B2.5 mAb binding to CD44 molecules both expressed by both T cells and monocytes. In the absence of monocytes, proliferation can be restored by solid-phase 8B2.5 mAb, or, to a lesser extent, by adding interleukin 2. Although CD3 and CD44 surface molecules are found physically independent, T cell activation via the CD44 pathway is inhibited by CD3 modulation. In addition to the direct role of CD44 molecules in T cell proliferation, CD44 mAb can up- or- down-regulate the CD3 and CD28 pathways, depending on the presence of monocytes. These results suggest that T cell and monocyte binding to high endothelial venule or extracellular matrix proteins could further promote clonal expansion of resting T cells migrating in certain specific anatomic sites.


Human endothelial cells transfected by SV40 T antigens: characterization and potential use as a source of normal endothelial factors.

A putative role for the vascular endothelium as target for autoantibodies has been suggested in several autoimmune disorders and connective-tissue diseases. However, there are some difficulties linked to the use of cultured endothelial cells (EC) that limit considerably the extensive studies on the nature of endothelial target antigens involved. To overcome this problem, human EC, derived from umbilical veins, were transfected with recombinant plasmid pSV1 which contained the early genes of simian virus SV40. These transfected cells, called EC-pSV1, are able to grow without EC growth supplement and demonstrate a population doubling time of about 50 h. Among the EC properties, EC-pSV1 retain intracellular content of angiotensin-converting enzyme activity, exhibit constitutive production of interleukin 6 and of a growth-promoting activity on early passage EV, express intercellular adhesion molecule 1 (ICAM-1) and its up-regulation by tumor necrosis factor alpha, but have lost the expression of factor VIII-related antigen. Moreover, EC-pSV1 express a 55-kDa antigen found on EC and human platelets, and presumably acting as an antibody target in some cases of non-allergic asthma. However, at the 50-55th generation, morphological changes and altered growth behavior were visible. This work demonstrates that transfection of EC with SV40 T antigens may be of interest, particularly in areas of research including the study of EC targets involved in different human diseases.


Major histocompatibility complex (MHC) control of CD4 T cell subset activation. II. A single peptide induces either humoral or cell-mediated responses in mice of distinct MHC genotype.

CD4 T cells activated in vivo in response to human collagen type IV (hCol IV) resemble either T helper type 1 (Th1) or Th2 cells depending on the major histocompatibility complex (MHC) class II genotype of the responding mice. H-2s mice were shown to selectively activate Th1-like cells, releasing interleukin (IL 2 and interferon-gamma in response to hCol IV, whereas H-2b.d mice were shown to selectively activate Th2-like cells, releasing IL 4 and IL 5 in response to hCol IV. These results suggested that MHC class II regulated the type of effector function observed during an immune response. It was of interest to determine if the functional difference observed between the CD4 T cells of the two strains was due to the presentation of different peptides of the hCol IV molecule by the two MHC class II molecules. The present results demonstrate that a single peptide of the collagen IV molecule will elicit a Th1-like response in H-2s strains and Th2-like responses in H-2b.d strains, as was observed when using the intact hCol IV molecule. Furthermore, the failure to generate Th1-like responses in H-2b.d could be overcome by increasing the dose of this peptide in vitro. Compared to H-2s, the Th1-like response in H-2b required 100 times the amount of peptide to reelicit an equivalent response. These data suggest that a single peptide of hCol IV can control the type of effector response observed.


Protection from lethal graft-vs.-host disease by donor stem cell repopulation.

Graft-vs.-host reaction (GVHR) induced in non-irradiated F1 mice with DBA/2J parental spleen and lymph node (LN) cells usually does not lead to acute GVH disease (GVHD). This contrasts with the GVHR induced in other parent-F1 combinations involving both major histocompatibility complex (MHC) class I and class II differences between donor and host. Most signs of acute GVHD in non-irradiated F1 mice relate to immunodeficiency following destruction of the lymphohemopoietic system of the host, which leads to wasting and death due to infections. This sequence of events is prevented when donor lymphoid cells, originating from grafted stem cells, repopulate the destroyed lymphohemopoietic system of the host. To examine whether a "silent" repopulation of the F1 host by donor stem cells might underly the absence of clinical signs of acute GVHD when GVHR is induced with DBA/2J lymphoid cells, GVHR was induced with LN cells, which do not contain stem cells. Indeed, GVHR induced in (C57BL/10 x DBA/2J)F1 (BDF1) mice with 80 x 10(6) DBA/2J LN cells led to acute GVHD. Signs of acute GVHD such as wasting and death did not occur when donor stem cells, from an inoculum of DBA/2J spleen and LN cells, were allowed to repopulate the lymphohemopoietic system of the host. The effect of donor stem cells on clinical signs of acute GVHD was more apparent when (B10.D2 x DBA/2J)F1, instead of DBA/2J, lymphoid cells were used to induce GVHR. The detection of alloreactive anti-host cytotoxic T lymphocyte (CTL) activity during acute GVHD induced with DBA/2J donor lymphoid cells supports the hypothesis that such CTL contribute to the destruction of the host immune system in acute GVHD.


Differential sensitivity of freshly isolated and cultured murine Langerhans cells to ultraviolet B radiation and chemical fixation.

Previous studies have demonstrated that low doses of ultraviolet B (UVB) radiation (100 J/m2) abrogate the accessory function of freshly isolated murine epidermal Langerhans cells (fLC) and cause a parallel decrease in the ability of LC to express increased amounts of ICAM-1 (CD54) in vitro. We have subsequently observed that the accessory cell function of cultured LC (cLC), as assessed by their ability to support anti-CD3 monoclonal antibody (mAb)-induced T cell mitogenesis, was not inhibited by levels of UVB exposure (100 J/m2) that completely inhibited the function of fLC, although exposure of cLC to UVB radiation (100 J/m2) resulted in a decrease in the level of ICAM-1 expression on most cLC and a concomitant decrease in cLC survival during a subsequent 24-h incubation. Time course studies revealed that T cells stimulated with anti-CD3 mAb in the presence of cLC became committed to proliferate 4-8 h after culture initiation, while 24-30 h of co-culture was required for irreversible T cell activation when fLC were utilized as accessory cells. In addition, paraformaldehyde (PFA)-fixed (non-viable) cLC supported anti-CD3 mAb-induced T cell proliferation, whereas PFA-fixed fLC were ineffective. We propose that cLC are functionally resistant to low doses of UVB radiation and chemical fixation because cLC express sufficient levels of the adhesion or co-stimulatory molecules [including ICAM-1 and Mac-1 (CD11b/CD18)] required to induce T cell activation. Conversely, fLC are sensitive to the effects of UVB radiation and chemical fixation because these physicochemical agents prevent acquisition of critically important surface molecules in culture.


A method for quantitating motor deficits in a nonhuman primate following MPTP-induced hemiparkinsonism and co-grafting.

This report describes a nonhuman primate model of MPTP-induced hemiparkinsonism and the recovery of motor function following co-grafting of adrenal medullary tissue and peripheral nerve into the lesioned area of the brain. A rhesus monkey (Macaca mulatta) trained to perform a complex, discrete-trial, operant task served as the subject. After behavioral performance on the task had stabilized and a high level of accuracy was maintained, 0.4 mg/kg MPTP was infused acutely via the left carotid artery to produce a marked impairment of movement of the right arm. Eighteen weeks later, medullary tissue from the left adrenal gland was grafted along with peripheral nerve into the left caudate nucleus. During the original baseline training condition, right- and left-hand performances were comparable on all dependent measures. However, right-hand performance was severely impaired following unilateral MPTP treatment, and left-hand performance was unaffected. Right-hand performance recovered only after adrenal medullary tissue was transplanted with peripheral nerve into the brain. Neuroanatomical analysis of brain tissue showed the anticipated neuronal loss in the left substantia nigra due to MPTP administration and evidence of adrenal medullary cell survival in the area of the co-graft. The data demonstrate that the rhesus monkey and the behavioral task developed during this study can be efficacious in characterizing the effects of MPTP on psychomotor function and in assessing the outcome of new strategies for treating Parkinson's disease.


Genetically tailored atrial natriuretic factor-dependent guanylate cyclase. Immunological and functional identity with 180 kDa membrane guanylate cyclase and ATP signaling site.

Biochemical and immunological studies have established that one of the signal transducers of atrial natriuretic factor (ANF) is a 180 kDa membrane guanylate cyclase (180 kDa mGC), which is also an ANF receptor; obligatory in the transduction process is an intervening ATP-regulated step, but its mechanism is not known. GC alpha is a newly discovered member of the guanylate cyclase family whose activity is independent of the known natriuretic peptides, and the enzyme is not an ANF receptor. The genetically tailored GC alpha, GC alpha-DmutGln338Leu364, however, is not only a guanylate cyclase but also an ANF receptor and is structurally and functionally identical to the cloned wild-type ANF receptor guanylate cyclase, GC-A. We now report that the ANF-dependent guanylate cyclase activity in the particulate fractions of cells transfected with GC alpha-DmutGln338Leu364 was inhibited by the 180 kDa mGC polyclonal antibody, and with this antibody probe it was possible to purify the 130 kDa expressed receptor; the hormone-dependent cyclase activity of this receptor was exclusively dependent upon ATP; and through site-directed mutational studies with GC alpha mutants, the signaling sequence that defines ATP binding site was identified. We thus conclude that 180 kDa mGC and the mutant protein are immunologically similar, both proteins are linked to the ANF signal in the generation of cyclic GMP synthesis; and in both the ligand binding and catalytic activities are bridged through a defined ATP binding module.


PKC-independent inhibition of glutamate exocytosis by arachidonic acid in rat cerebrocortical synaptosomes.

In rat cerebrocortical synaptosomes, the addition of 4 beta-phorbol dibutyrate (4 beta-PDBu) and arachidonic acid enhances and decreases, respectively, the glutamate release evoked by 4-aminopyridine. Pretreatment of synaptosomes with 12-O-tetradecanoylphorbol 13-acetate (TPA) or pre-incubation with staurosporine, prevent the stimulatory effect of 4 beta-PDBu, but are without effect on the inhibitory action of arachidonic acid. Moreover, methyl arachidonate, which is not effective as a PKC activator, also strongly inhibits glutamate exocytosis. These results suggest that PKC is not involved in the inhibition of glutamate release by arachidonic acid.


Thy-1 involvement in neurite outgrowth: perturbation by antibodies, phospholipase C, and mutation.

Thy-1 is a major cell surface protein anchored in the plasma membrane of neurons and lymphocytes by a covalent glyco-phosphatidyl-inositide linkage. Despite thorough characterization of the molecule's physicochemical properties, its biological function remains elusive. In this study we demonstrate that (i) monoclonal antibodies directed against Thy-1 are capable of enhancing neurite outgrowth from sympathetic neurons in culture, as well as stimulating the initiation of neurite sprouting from cultured adrenal chromaffin cells and PC12 cells. This effect is not observed with monovalent, Fab antibody fragments. Treatment with intact antibodies also results in the shedding of Thy-1 into the culture medium. (ii) Treatment of chromaffin cells with phosphatidyl-inositol-specific phospholipase C also results in an induction of neurite sprouting. The lipase effect can be blocked by preincubating the cells with monovalent anti-Thy-1 Fab fragments, indicating that the outgrowth stimulation is specifically due to removal of Thy-1. (iii) An entirely different approach to elucidating the function of Thy-1 involves mutagenesis of PC12 cells. Selection for Thy-1-deficient mutants revealed that cells lacking Thy-1 sprout neurites spontaneously at a very high frequency. A novel role for Thy-1 is proposed wherein the results of the mutant cell studies are compatible with the antibody and lipase data. Each of the perturbations can be viewed as releasing an inhibition that Thy-1 normally exerts on neurite outgrowth. We suggest that Thy-1 normally acts to stabilize neuronal membranes and processes, possibly through homophilic interactions.


Thy-1 multimerization is correlated with neurite outgrowth.

Thy-1 is abundantly expressed in the vertebrate nervous system. Perturbation studies in vitro suggest that Thy-1 inhibits neurite outgrowth and stabilizes neuronal processes (N. K. Mahanthappa and P. H. Patterson. (1992). Thy-1 involvement in neurite outgrowth: Perturbation by antibodies, phospholipase C, and mutation. Dev. Biol. 150,47-59). We here report that Thy-1 participates in several types of homophilic interactions, each with differential sensitivity to reduction and boiling. The relative abundance of the multimeric forms of Thy-1 vary with the cell's ability to sprout neurites. Gel filtration chromatography of sympathetic neuron and PC12 cell lysates reveals that Thy-1 immunoreactivity appears in 25-, 45-, and 150-kDa forms. In neurons, Thy-1 immunoreactivity is distributed equally in all three forms, whereas in PC12 cells, the majority of Thy-1 immunoreactivity is found in the higher molecular weight forms. When PC12 cells are induced to sprout neurites with NGF, the Thy-1 size distribution becomes identical to that of neurons. The three forms of Thy-1 immunoreactivity are likely to be homomultimers of Thy-1 because immunoaffinity-purified, soluble Thy-1 also forms complexes similar in size to those found in neuronal extracts. To test whether Thy-1 multimerization may occur through interactions like those between immunoglobulin heavy and light chains, synthetic peptides corresponding to candidate sites for such associations in Thy-1 were tested for their effects on multimerization and neurite outgrowth. One peptide increases the amount of monomeric Thy-1 relative to total Thy-1, and promotes outgrowth. These results suggest that multimeric forms of Thy-1 inhibit process outgrowth and neurite sprouting by stabilizing the surface membrane and/or underlying cytoskeleton.


Role of Glu318 and Thr319 in the catalytic function of cytochrome P450d (P4501A2): effects of mutations on the methanol hydroxylation.

Polar amino acids in the (putative) distal site are well conserved in P450s. For example, Glu318 for P450d is well conserved as either Glu or Asp for P450s, and Thr319 for P450d is also conserved for P450s. We have studied how mutations at Glu318 and Thr319 of P450d influence the catalytic activity toward methanol associated with the activation of O2. Catalytic activities of Glu318Asp, Glu318Ala, and Thr319Ala mutants toward methanol were 60, 25, and 38%, respectively, compared with that of the wild type. O2 consumption and NADPH oxidation rates of each mutants varied corresponding to the catalytic activities. However, surprisingly, efficiency (16-40%) of incorporated O to the substrate vs. consumed O2 for the Glu318Ala and Thr319Ala mutants were higher than that (9%) of the wild type. In addition, H2O2, which is produced from uncoupling for the wild-type P450d, was not observed for reaction of the Glu318Ala and Thr319Ala mutants. It seemed that consumed O2 was partially reduced to 2 mol of H2O by 4-electron transfer from NADPH for the wild-type and Thr319Ala mutant. However, for the two Glu318 mutants, it appeared that the consumed O2 was not reduced in the same way. It was thus suggested that the conserved Glu318 and Thr319 of P450d are not essential for the activation of O2 in the methanol oxidation. Role of the water molecule or the methanol molecule in the catalytic function was implied.


[Comparative effects of ricinoleic acid and senna on orocecal and oroanal transit time in healthy subjects. Application of the salacylazosulfapyridine method].

The appearance in plasma of sulphapyridine after oral administration of salicylazosulphapyridine (Salazopyrin) was shown to be useful for measuring the orocecal transit time in normal subjects. The purpose of this study was to use this method in diarrhea with accelerated intestinal transit time. A two-step study was performed in 12 healthy volunteers: a) under resting conditions; b) 2 weeks later with ricinoleic acid 40 ml (n = 6) or senna 19 mg (X-Prep = 1.2 g; n = 6) administration. In each step, Salazopyrin (2 g) and 20 radiopaque markers were ingested with a 200 kcal meal (Polydiet TCM = 200 ml). The following parameters were determined: a) plasmatic level of sulphapyridine (spectrophotometry) at 30 min intervals during 12 h; b) 2-day stool frequency and weight; c) oro-anal transit time (passage of the first marker and half of the markers in stools). In one subject, no sulphapyridine level was detected after administration of ricinoleic acid. With senna, 2 day stool frequency and weight increased by 80 and 131 percent respectively: orocecal transit time decreased from 6.1 +/- 1.3 to 4.8 +/- 1.2 h (m +/- SD; P less than 0.01) and oro-anal transit time (first marker) decreased from 31.8 +/- 9.6 to 20.7 +/- 8.9 (P less than 0.05). With ricinoleic acid, 2 day stool frequency and weight increased by 212 and 350 percent respectively; orocecal transit time decreased from 5.8 +/- 1.8 to 2.2 +/- 0.7 (P less than 0.01) and oroanal transit time (first marker) decreased from 25.3 +/- 7.1 to 8.0 +/- 6.8 h (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Benefit of resection of metastatic gastrinoma in multiple endocrine neoplasia type I.

Although gastrinoma resection is generally advocated for patients with the sporadic form of nonmetastatic Zollinger-Ellison syndrome, there is controversy regarding the surgical management of the gastrinoma among patients with multiple endocrine neoplasia type I (MEN-I). Using strict criteria, to date no biochemical cures of the Zollinger-Ellison syndrome lasting greater than 5 months have been achieved by gastrinoma resection among patients with MEN-I. Whereas resections of hepatic metastases have been performed in patients with sporadic gastrinoma, none have been reported among patients with MEN-I. The current report describes a patient with MEN-I, closely followed up for 30 years, in whom enlargement of pancreatic gastrinoma and development of hepatic gastrinoma was observed to occur over 3 years. After preoperative localization, an 80% pancreatectomy and a left lateral segmentectomy of the liver were performed. Sixteen months after the operation, secretin and calcium provocative testing showed that the patient's fasting gastrin and stimulated plasma gastrin concentrations were normal; also, results of computerized tomographic angiography, selective abdominal angiography, and hepatic venous sampling for gastrin after intra-arterial secretin injection were negative for gastrinoma. By achieving a 16-month cure of gastrinoma, this case shows that an aggressive surgical approach can benefit certain patients with gastrinoma who have MEN-I even in the presence of hepatic metastases.


DNA patterns of Helicobacter pylori isolated from gastric antrum, body, and duodenum.

Biopsy specimens for culture of Helicobacter pylori were obtained from two different sites in the antrum, gastric body, and duodenal cap in 20 patients during endoscopic investigation of dyspepsia. H. pylori was identified in 64 isolates obtained from 15 of the 20 patients. Analysis of chromosomal DNA from these isolates of H. pylori showed that 13 of 15 patients harbored a single strain of H. pylori throughout their stomach and duodenum. Two differing H. pylori types were found in two patients. Unique DNA patterns were shown in each of the 15 patients. The genetic heterogeneity of H. pylori is unexplained but it could be of considerable value for epidemiological studies.


Escherichia coli enterotoxin (STa) binds to receptors, stimulates guanyl cyclase, and impairs absorption in rat colon.

To determine the contribution of the colon in Escherichia coli heat-stable enterotoxin-mediated diarrheal disease, toxin binding, guanyl cyclase activation, and toxin-induced water flux in the rat colon and ileum were compared. Scatchard analysis suggested a single class of heat-stable enterotoxin receptors with an affinity constant of binding of 10(9) L/mol in both colonocytes and ileocytes; however, the number of toxin receptors per cell was 3.5-fold greater in coloncytes than ileocytes (8.32 +/- 1.33 x 10(5) vs. 2.33 +/- 0.28 x 10(5) receptors per cell; P = 0.02). Heat-stable enterotoxin stimulated guanyl cyclase activation in an identical dose-dependent manner in proximal colonic and ileal membranes, with similar sensitivity and maximum response. Heat-stable enterotoxin also inhibited net water flux to a similar degree in both colon and ileum (-47.8 vs. -48.4 microL.cm-1.h-1, respectively) at a dose of 8 nmol/L. At this dose in the colon, because of a higher baseline of absorption, absorption continued, but at a diminished level. At this dose in the ileum, heat-stable enterotoxin induced net secretion. These data are consistent with the concept that heat-stable enterotoxin-induced diarrheal disease results from a decreased absorptive capacity in the colon in the face of increased small intestinal fluid secretion.


Arginine vasopressin inhibits phasic contractions and stimulates giant contractions in monkey colon.

Abdominal cramps and urgent defecation are common side effects of clinical doses of arginine vasopressin, indicating that the drug may have stimulating effects on colonic motor activity. Four strain-gauge transducers were implanted on the colon in six monkeys. A blood flow probe was fixed on the inferior mesenteric artery. After a 1-hour control recording, vasopressin, 0.13, 1.3, or 13.0 ng.kg-1.min-1, was infused intravenously for 90 minutes. The frequency of basal colonic contractions was reduced with increasing doses of vasopressin, but their mean amplitude and duration were not altered. Giant migrating contractions associated with defecation were initiated by the highest dose of vasopressin. Atropine had no effect on these giant migrating contractions but completely inhibited normal phasic contractions. Hexamethonium completely inhibited both giant migrating contractions and phasic contractions. Parasympathetic denervation of the colon did not inhibit giant migrating contractions initiated by vasopressin. Our findings suggest that the physiological concentrations of serum vasopressin present perioperatively may transiently inhibit spontaneous colon contractions but are unlikely to be the major cause of postoperative ileus. The giant migrating contractions initiated by vasopressin may account for the defecation associated with pharmacological doses of vasopressin. The initiation of giant migrating contractions by vasopressin may be mediated through a neural pathway.


The function of Gp170, the multidrug-resistance gene product, in the brush border of rat intestinal mucosa.

Gp170 is a transmembrane glycoprotein that is overexpressed in multidrug-resistant tumor cells and is present in the apical plasma membrane domain of small intestinal mucosal cells. The function of Gp170 was studied in the small intestine of the rat. Jejunal and ileal brush border membrane vesicles, but not basolateral membrane vesicles, manifested adenosine triphosphate (ATP)-dependent transport of daunomycin, a substrate for Gp170, and contained a approximately 170-kilodalton protein that reacts with anti-Gp170 monoclonal antibody. Whereas ATP supported daunomycin transport, nonhydrolyzable ATP analogues were ineffective. ATP-dependent daunomycin transport by brush border vesicles was unidirectional (inside to outside) and temperature dependent and was blocked by Gp170 inhibitors but not by taurocholate or bromsulphalein glutathione. Studies using everted small intestine revealed transport of rhodamine 123, a Gp170 substrate, from the serosal surface through the mucosa and inhibition by Gp170 inhibitors. These results suggest that Gp170 in rat small intestinal brush border membrane vesicles is an ATP-dependent efflux pump responsible for the transport of Gp170 substrates into the small intestinal lumen. Gp170 may protect against exogenously derived potentially damaging hydrophobic cations and contribute to the rarity of small intestinal cancer in humans and many animals.


Altered glutamine metabolism in rat portal drained viscera and hindquarter during hyperammonemia.

In normal rats, muscle is the major glutamine releasing organ and gut is the major glutamine consuming organ. It has been suggested that enhanced muscle ammonia detoxification and gut ammonia production occurs during liver insufficiency-induced hyperammonemia. Therefore, ammonia and amino acid fluxes across portal-drained viscera and hindquarter, and muscle concentrations were measured in portacaval shunted and acute liver ischemia rats. Arterial ammonia and most amino acids were increased after portacaval shunting and increased progressively during liver ischemia, but net hindquarter ammonia uptake was not observed. Net hindquarter glutamine efflux was increased during portacaval shunting, but it decreased during liver ischemia, while muscle glutamine concentrations increased. The comparable net portal drained viscera glutamine uptake in normal and portacaval shunted rats changed during liver ischemia from net uptake to release, coinciding with release of most other amino acids. These results cast doubt on the ammonia detoxifying role of muscle during acute liver ischemia-induced hyperammonemia in the rat. The portal drained viscera glutamine release during severe hyperammonemia could be due to intestinal damage.


Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain.

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13,472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3'-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5'-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.


Correlation of L-myc RFLP with metastasis, prognosis and multiple cancer in lung-cancer patients.

For further study of the correlation of L-myc restriction-fragment-length polymorphism (RFLP) and metastasis of lung cancer to lymph nodes or other organs at the time of surgery, L-myc RFLP was analyzed in 252 Japanese lung-cancer patients. A close correlation between L-myc RFLP and metastasis was confirmed in this large number of patients (p = 0.01). The correlation was particularly pronounced in cases of adenocarcinoma and squamous-cell carcinoma. Poor prognosis (additional metastases after surgery) was observed in lung-cancer patients with L-S (identified as long and short bands produced with EcoRI) and S-S type L-myc RFLP. In addition, the death rate of lung-cancer patients with the L-S and S-S types was greater than that of those with the L-L type. Lung-cancer patients of the L-S and S-S types had almost 4 times higher incidence of multiple cancer in the lung, pharynx and other organs than those with the L-L type. Our results indicate that, in patients with lung cancer, genetic disposition with respect to the L-myc gene influences the extent of metastasis, the incidence of multiple cancers and prognosis.


SDZ PSC 833, a non-immunosuppressive cyclosporine: its potency in overcoming P-glycoprotein-mediated multidrug resistance of murine leukemia.

Cyclosporin A (CsA, Sandimmune) is known to reverse P-glycoprotein (P-gp170)-mediated multidrug resistance as efficiently as other prototype compounds of resistance modifiers. The immunosuppressive activity and nephrotoxicity of CsA, however, may limit its clinical use. PSC-833, a new cyclosporine, exerts a similar resistance-modifying activity but lacks toxicity or immunosuppressive activity. We have tested its potency in vitro and in vivo on the L1210 leukemia cell line transfected with a full-length cDNA copy of the human mdr I gene, which showed a stable 30-fold resistance towards adriamycin as compared to the parental cell line. In vitro growth of the transfected cell was unchanged. In vivo growth was less aggressive; the survival time of inoculated mice was prolonged. In vitro, PSC-833 was at least as potent as CsA or verapamil in reversing multidrug resistance. In vivo, the drug-resistant L1210 leukemia was completely unresponsive to i.v. monotherapy with adriamycin at its maximum tolerated dose (MTD). PSC-833 enhanced the activity and toxicity of adriamycin. The MTD of adriamycin was about 3 times lower than when given alone. On this basis, the MTD of i.v. adriamycin in combination with oral PSC-833 successfully overcame refractoriness to treatment. Survival times of the mice were considerably prolonged and even some cures of leukemic mice occurred.


Imipramine treatment of children with separation anxiety disorder.

The efficacy of imipramine was investigated in 20 children (ages 6 to 15) with separation anxiety disorder. Children were treated for a month with vigorous behavioral treatment. If they did not respond, they entered a double-blind, randomized, 6-week trial of imipramine or placebo. Of 45 children accepted, 21 (47%) entered the trial. About half the children improved with either treatment, and no superiority for imipramine was obtained. There was no instance of clinically significant EKG changes. This small study failed to replicate previous findings of imipramine efficacy in a similar, but larger, clinical population.


Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae.

Extracellular muramidase-2 of Enterococcus hirae ATCC 9790 was purified to homogeneity by substrate binding, guanidine-HCl extraction, and reversed-phase chromatography. A monoclonal antibody, 2F8, which specifically recognizes muramidase-2, was used to screen a genomic library of E. hirae ATCC 9790 DNA in bacteriophage lambda gt11. A positive phage clone containing a 4.5-kb DNA insert was isolated and analyzed. The EcoRI-digested 4.5-kb fragment was cut into 2.3-, 1.0-, and 1.5-kb pieces by using restriction enzymes KpnI, Sau3AI, and PstI, and each fragment was subcloned into plasmid pJDC9 or pUC19. The nucleotide sequence of each subclone was determined. The sequence data indicated an open reading frame encoding a polypeptide of 666 amino acid residues, with a calculated molecular mass of 70,678 Da. The first 24 N-terminal amino acids of purified extracellular muramidase-2 were in very good agreement with the deduced amino acid sequence after a 49-amino-acid putative signal sequence. Analysis of the deduced amino acid sequence showed the presence at the C-terminal region of the protein of six highly homologous repeat units separated by nonhomologous intervening sequences that are highly enriched in serine and threonine. The overall sequence showed a high degree of homology with a recently cloned Streptococcus faecalis autolysin.


P-glycoprotein. ATP hydrolysis by the N-terminal nucleotide-binding domain.

Two ATP-binding domains are found in members of the family of ATP-dependent transport proteins, which includes P-glycoprotein and cystic fibrosis transmembrane conductance regulator. To investigate the involvement of the two ATP-binding domains in the ATPase activity of P-glycoprotein, full-length and the 5'-half of human MDR1 cDNA, which encodes P-glycoprotein, were fused with the Escherichia coli lacZ gene and expressed in NIH3T3 cells. Immunoprecipitated full-length P-glycoprotein beta-galactosidase showed ATPase activity with apparent specific activity of 180 nmol/mg/min, a value higher than previously reported, in the presence of phospholipids, suggesting that stabilization of the transmembrane domains is necessary for ATP hydrolysis. N-terminal half P-glycoprotein-beta-galactosidase also showed ability to hydrolyze ATP but with slightly lower specific activity. Both ATPase activities showed similar characteristics when the effect of several inhibitors was analyzed, indicating that the N-terminal ATP-binding domain contains all residues necessary to hydrolyze ATP without interacting with the C-terminal ATP-binding domain.


Tyrosine phosphatase inhibition permits analysis of signal transduction complexes in p185HER2/neu-overexpressing human tumor cells.

The HER2/neu gene encodes a receptor tyrosine kinase that is highly homologous to the epidermal growth factor receptor. Overexpression of the receptor in mammary and ovarian carcinoma correlates with poor patient prognosis. To determine how the overexpression of a normal receptor leads to the generation of an oncogenic signal, we compared the patterns of tyrosine phosphorylation in tumor-derived human cell lines expressing high levels of p185HER2/neu. In intact SKBR3 cells, basal phosphorylation of p185HER2/neu was not detected. However, pretreatment of cells with the tyrosine phosphatase inhibitor, sodium orthovanadate, led to the detection of phosphotyrosine on phospholipase C-gamma (PLC-gamma), GTPase-activating protein but not on the RAF-1 kinase. Strikingly, PLC-gamma was detected in a complex which contained multiple tyrosine-phosphorylated polypeptides. This complex was detected only in cytoplasmic fractions and had a distinct composition in different p185HER2/neu-overexpressing cell lines. Although GTPase-activating protein has been found previously in association with proteins of 190 and 62 kDa in fibroblasts, in SKBR3 cells it was found associated with multiple additional tyrosine-phosphorylated polypeptides. These experiments show that SKBR3 cells possess high levels of protein tyrosine phosphatase that can act upon p185HER2/neu. Moreover, they reveal, for the first time, the presence of PLC-gamma and GTPase-activating protein in cytosolic complexes containing a variety of other tyrosine-phosphorylated polypeptides. These observations suggest novel possibilities for the specific definition of receptor-generated signals in tumor cells.


Dipeptidyl peptidase IV (CD 26) gene expression in enterocyte-like colon cancer cell lines HT-29 and Caco-2. Cloning of the complete human coding sequence and changes of dipeptidyl peptidase IV mRNA levels during cell differentiation.

A cDNA (DPCR1) specific for human intestinal dipeptidyl peptidase IV (DPP IV) has been isolated. This 1.7-kilobase cDNA, together with a previously published partial sequence, covers the entire open reading frame of human DPP IV plus 67 base pairs of the 3'-untranslated end. Human DPP IV is a 766-amino acid polypeptide with a high degree of homology with the rat liver protein. The characterization of this molecular probe allowed us to definitively confirm the identity of DPP IV with CD 26, a mouse thymocyte activation antigen, a conclusion strengthened by the fact that we observed identical patterns on Southern blot of human genomic DNA hybridized either with human DPP IV or mouse CD 26 cDNA probe. Using this new tool, we have investigated the expression of DPP IV during the onset of enterocytic differentiation of two cultured human colon cancer cell lines, HT-29 and Caco-2. Whatever the cell line and the culture conditions, DPP IV expression strictly correlates with the presence of a differentiated phenotype, as shown by enzyme activity and the steady state amount of the protein measured by indirect immunofluorescence and Western blot. Accordingly, DPP IV biosynthesis exclusively increases in cells that display an enterocytic differentiation. Neither the glycosylation nor the stability of the protein appear to be dependent on the state of enterocytic differentiation. The DPP IV mRNA level remains very low in undifferentiated cell populations and specifically increases in cells that undergo an enterocytic differentiation. These results strongly suggest that DPP IV gene expression is controlled at the transcriptional or posttranscriptional level during intestinal differentiation.


Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase.

Drug-resistant tumor cells actively extrude a variety of chemotherapeutic agents by the action of the multi-drug resistance (MDR1) gene product, the plasma membrane P-glycoprotein. In this report we show that the expression of the human MDR1 gene in cultured Sf9 insect cells via a baculovirus vector generates a high activity vanadate-sensitive membrane ATPase. This ATPase is markedly stimulated by drugs known to interact with the P-glycoprotein, such as vinblastine and verapamil, and the ability of the various drugs to stimulate the ATPase corresponds to their previously observed affinity for this transporter. The drug-stimulated ATPase is not present in uninfected or mock-infected Sf9 cells, and its appearance correlates with the appearance of the MDR1 gene product detected with a monoclonal anti-MDR protein antibody and by labeling with 8-azido-ATP. The drug-induced ATPase requires magnesium ions, does not utilize ADP or AMP as substrates, exhibits a half-maximal activation at about 0.5 mM MgATP, and its maximal activity (about 3-5 mumol/mg MDR protein/min) approaches that of the well characterized ion transport ATPases. These results provide the first direct demonstration of a high capacity drug-stimulated ATPase activity of the human multidrug resistance protein and offer a new and simple assay for the investigation of functional interactions of various drugs with this clinically important enzyme.


Glutamine and glutamate metabolism in normal and heat shock conditions in Drosophila Kc cells: conditions supporting glutamine synthesis maximize heat shock polypeptide expression.

We have previously reported that Drosophila Kc cells require glutamine for maximal expression of heat shock proteins in stressed conditions (Sanders and Kon: J. Cell. Physiol. 146:180-190, 1991). The mechanism of this effect has been investigated by comparing the metabolic utilization of glutamine in conditions which support hsp expression with that of glutamate in conditions where up to 100-fold less hsp is synthesized. This comparison showed that free ammonia was generated by cells incubated in the presence of glutamine in 37 degrees C (heat shock) conditions, but not at 25 degrees C, and not in the presence of glutamate in either normal or heat shock conditions. There was no difference in the amount of [14C]O2 generated from either [14C]-labeled amino acid in the tricarboxylic acid cycle, but three- to four-fold more alanine was synthesized in cells incubated in glutamine than in glutamate. Treating the cells with aminotransferase inhibitors to artificially increase NH3 release raised hsp expression in the presence of glutamate to maximal levels characteristic of glutamine. This potentiation correlated with inhibition of alanine aminotransferase. Since only NH3 production correlated with hsp expression in heat shock conditions in the presence of glutamine, and NH3 addition to glutamate also resulted in maximal hsp expression, we measured glutamine production in glutamate plus NH3 and observed net glutamine synthesis. The supposition that glutamine itself is responsible for the regulatory changes supporting maximal hsp expression was supported by the finding that the glutamine analog, 6-diazo-5-oxo-L-norleucine (DON), mimicked the effects of glutamine. We conclude that glutamine imposes regulatory changes which alter nitrogen metabolism and support hsp expression in Kc cells.


Sensitivity and specificity of a recombinant transmembrane glycoprotein (rgp21)-spiked western immunoblot for serological confirmation of human T-cell lymphotropic virus type I and type II infections.

Serum specimens (n = 2,712) obtained from individuals residing in diverse geographic regions and categorized as seropositive (n = 122), seroindeterminate (n = 523), or seronegative (n = 2,067) for human T-cell lymphotropic virus (HTLV) infection in accordance with U.S. Public Health Service guidelines were retested by recombinant transmembrane protein (rgp21)-spiked Western immunoblotting. Of the 122 HTLV-positive specimens, those from 85 of 85 (100%) U.S. blood donors, 2 of 2 (100%) Brazilians, 1 of 2 (50%) Indonesians, 14 of 14 (100%) Solomon Islanders, and 18 of 19 (95%) Papua New Guineans reacted with rgp21, yielding an overall sensitivity of 98% (120 of 122). Specimens from individuals whose infections were confirmed to be HTLV type I or HTLV type II by the polymerase chain reaction assay reacted equally well with rgp21. Of the 523 HTLV-indeterminate specimens, those from 21 of 379 (5.5%) U.S. blood donors, 3 of 6 (50%) Brazilians, 10 of 23 (44%) Ugandans, 8 of 49 (16%) Indonesians, 4 of 36 (11%) Solomon Islanders, and 5 of 30 (17%) Papua New Guineans reacted with rgp21. None of these 51 specimens reacted with native gp46 and/or gp61/68 in a radioimmunoprecipitation assay, suggesting a false-positive reaction (9.75%). Of the 2,067 HTLV-negative specimens, 12 reacted with rgp21, yielding a false-positivity rate of 0.6%. These data indicate that while detection of rgp21 is highly sensitive, it can yield false-positive results. Thus, specimens exhibiting reactivity with rgp21 in the absence of reactivity with native gp46 and/or gp61/68 by Western blot should be tested further by a radioimmunoprecipitation assay to verify HTLV type I or type II infection.


Evidence for functional heterogeneity of rat CD4+ T cells in vivo. Differential expression of IL-2 and IL-4 mRNA in recipients of cardiac allografts.

The in vivo relevance of functional dichotomy of CD4+ Th clones was studied by analyzing the induction of mRNA encoding for Th1- (IL-2) and Th2- (IL-4) specific lymphokines in a model of accelerated (24 h) cardiac allograft (Tx) rejection in presensitized rats. The polymerase chain reaction-assisted screening of total cellular RNA from cardiac Tx of otherwise untreated sensitized recipients has revealed sequential lymphokine mRNA expression, with the peak of IL-2 mRNA (6-12 h) preceding that for IL-4 mRNA, which was maximal at the time of actual Tx loss (24 h). Both IL-2 and IL-4 transcripts could be readily detected by polymerase chain reaction analysis in the spleens during the course of accelerated rejection. Treatment of prospective cardiac Tx recipients with BWH-4, a mouse anti-rat CD4 mAb, abrogated rejection at 24 h and prolonged cardiac Tx survival to ca. 11 days, coinciding with significantly diminished IL-2 mRNA expression. In contrast, CD4 targeted therapy preserved intra-Tx and splenic transcription of the IL-4 gene. Spleen lymphocytes from mAb-conditioned recipients separated by magnetic microspheres into phenotypically distinct subpopulations, showed differential induction of IL-2 and IL-4 mRNA. Thus, IL-2 mRNA was at most very weakly expressed, whereas IL-4 transcription was strongly induced both in CD4+ T cells and its OX-22- subset. This study demonstrates the induction of IL-4 mRNA in situ in the rat system, describes discordant elaboration of IL-2 and IL-4 mRNA in untreated/anti-CD4 mAb-treated cardiac Tx recipients, and identifies OX-22- CD4+ T cells as the IL-4 mRNA producers. Thus, these results provide evidence for functional heterogeneity of rat CD4+ T cells in vivo, as defined by divergent mRNA lymphokine transcription profiles.


Identification of IL-7-dependent bone marrow-derived Thy-1-B220- lymphoid cell clones that rearrange and express both Ig and T cell receptor genes.

Bone marrow stromal cell lines and lymphoid cell lines were co-established from the Whitlock-Witte type of long term liquid cultures of MRL/1 and C57BL/10 (B10) (Thy-1.1) bone marrow cells. The present study investigates the immunologic nature of parental and cloned lymphoid cell lines. Both strains of parental lines and their clones did not grow alone but proliferated on the monolayers of co-established parental stromal cell lines from a syngeneic or alternative strain. When various lymphokines or cytokines were tested for their capacity to support the growth of these lymphoid cell clones, only IL-7 could substitute for the growth-promoting function of stromal cells. These IL-7-dependent clones expressed neither Thy-1 nor B220 Ag. However, all of them from two strains were found to rearrange synchronously H chain of Ig as well as gamma chain of TCR genes. Some of the clones transcribed a mature size of IgH mRNA. Co-expression of mRNA for lambda 5 but not for IgL chain (kappa, lambda) genes resulted in the generation of cell surface mu chain in these clones. Other clones expressed a smaller size of IgH mRNA without exhibiting surface mu chain. Irrespective of the differences in IgH rearrangements and its mRNA expression, a mature size TCR gamma mRNA was detected in all of the clones. Thus, these results demonstrate the existence of untransformed (IL-7-dependent) immature lymphoid cells rearranging both Ig and TCR genes. Their unique features concerning cell surface markers (B220- mu+), specific growth factor requirement, and various modes of Ig/TCR gene rearrangements are discussed in the context of early lymphoid development.


Regulation of ICAM-1 expression and function in human dermal fibroblasts by IL-4.

ICAM-1 is found on the surface of many hematopoietic and nonhematopoietic cells and can function as an adhesive ligand for the integrin, leukocyte function-associated molecule-1 (LFA-1, CD11a/CD18). ICAM-1/leukocyte function-associated molecule-1 interaction has been shown to be of importance in many immune-mediated cell-cell adhesion reactions. In vitro, unstimulated human fibroblast cell cultures express low levels of ICAM-1. Using ELISA, cytofluorography, electron microscopy, Northern analysis, and an in vitro cell adherence assay, we demonstrate that treatment of human dermal fibroblasts with the cytokine IL-4 leads to an increase in cell surface ICAM-1 expression that is under transcriptional control as well as increased fibroblast adhesion to LFA-1-bearing T lymphocytes. The kinetics of increased ICAM-1 expression induced by IL-4 paralleled the increase in ICAM-1-dependent T lymphocyte adhesion. The increase in T cell adhesion was determined to be due to the effects of IL-4 on the fibroblasts and not the adhering T cells. Treatment of fibroblasts with IL-4 also resulted in enhanced binding of human rhinovirus, a recently reported additional ligand for ICAM-1. Virus binding was IL-4 dose dependent and could be inhibited with mAb to ICAM-1. Both the expression of ICAM-1 and the ICAM-1-dependent increase in T lymphocyte adhesion that was induced by IL-4 could be inhibited by preexposure of the fibroblasts to either IL-1 or IL-6, suggesting that multiple cytokines can have both positive and negative effects on human fibroblast ICAM-1 expression and function.


Pertussis toxin-induced lymphocytosis is associated with alterations in thymocyte subpopulations.

Pertussis toxin (Ptx), an important adjuvant for inducing certain organ-specific autoimmune diseases in mice, exerts multiple effects upon the immune system. In addition to its adjuvant effects, which include enhancement of delayed-type hypersensitivity and increased antibody production. Ptx elicits a marked lymphocytosis with a concomitant decrease in thymic weight. In vitro studies indicate that Ptx acts directly on thymocytes and that both susceptible and resistant populations exist. It is believed that these susceptible cells are released into the circulation and account, in part, for the T cell component of the lymphocytosis. We have used flow cytometry to analyze the CD4, CD8, and Thy-1 phenotypes of thymic and peripheral T cells from Ptx-treated mice. In the thymus, there is a dramatic decrease in the number of CD4+CD8+ (double positive) cells at all doses tested (0.25, 0.50, and 1.0 microgram) by day 4 after Ptx treatment. The double negative and single positive populations remain relatively constant. Analysis of Thy-1 expression reveals a significant reduction in Thy-1hi thymocytes, with little change in the Thy-1lo population. Thus Ptx primarily affects and depletes, in a dose-dependent fashion, thymic T cells with an immature phenotype. These results mimic those of corticosteroids, although neither prior adrenalectomy nor treatment with the antiglucocorticoid RU486 are able to prevent the effects of Ptx. In the periphery of Ptx-treated animals, the relative increase in the number of CD4+ T cells is more than that of CD8+ T cells. Double positive and Thy-1hi cells cannot be detected in appreciable numbers. These results are consistent with the concept that Ptx may drive immature thymocytes through accelerated maturation for release into the periphery as single positive, predominantly CD4+, Thy-1lo cells. Increased numbers of such cells may in part account for the immunopotentiating effects of Ptx, particularly as they relate to the induction of organ-specific autoimmune disease. Treatment with purified Ptx beta-oligomer fails to elicit any of the responses described above, indicating that the holotoxin is required for such activities.


A method of preparing blood leucocytes for flow cytometry which prevents upregulation of leucocyte integrins.

LeuCAM (CD11/CD18) cell-surface antigens are easily upregulated on cell manipulation ex vivo. A procedure for preparing leucocytes, in which human blood is immediately treated ex vivo with buffered formaldehyde and then the erythrocytes and platelets are removed by lysis and differential centrifugation, has been successfully applied to the analysis of LeuCAM antigen expression by flow cytometry. We show that the increased expression of monocyte CD11/CD18, which occurs when mononuclear leucocytes are separated by a standard Lymphoprep density gradient separation, can be avoided if cells are fixed immediately. Following this fixation polymorphs are unable to upregulate CD11/CD18 in response to fMLP stimulation in vitro. The technique produces lymphocyte, polymorph and monocyte populations that can be clearly defined on the basis of forward scatter and side scatter, and preserves the expression of various surface antigens; the percentages of gated lymphocytes expressing CD3, CD4, and CD8 were similar to those obtained using a commercial fixing and lysis solution. The processing does not render cells permeable to antibodies, as evidenced by our failure to stain cells with antibodies to intracellular antigens. We believed the method to be useful for measuring CD11/CD18 expression on blood leucocytes from normal or pathological specimens and to have application to the measurement of other cells surface antigens which may also be upregulated by the separation procedures.


Dehydroepiandrosterone as predictor for progression to AIDS in asymptomatic human immunodeficiency virus-infected men.

The steroid hormone dehydroepiandrosterone (DHEA) has been reported to protect against certain viral infections in animal models and to be a modest inhibitor of human immunodeficiency virus type 1 (HIV-1) infection in vitro. Serum DHEA levels were determined in 41 asymptomatic HIV-1-seropositive subjects, who progressed to AIDS within 5 years after entering a cohort study, in 41 HIV-1-seropositive controls, who remained asymptomatic, and in 41 HIV-1-seronegative controls. At entry, DHEA levels were higher in the seronegative group (median, 13.3 nmol/l) than in either the seropositive nonprogressors (median, 9.2 nmol/l; P = .01) or the progressors (median, 7.2 nmol/l; P less than .001). DHEA levels in the progressors approximately 5 months before the diagnosis of AIDS were lower than the levels in the nonprogressors after the same follow-up (median, 5.6 vs. 8.8 nmol/l; P = .007). DHEA levels less than 7 nmol/l and CD4+ cell counts less than 0.5 x 10(9)/l both proved to be independent predictors for disease progression in HIV-1-infected men.


Viral phenotype and immune response in primary human immunodeficiency virus type 1 infection.

Nineteen individuals were studied for virologic and immunologic events during primary human immunodeficiency virus type 1 (HIV-1) infection. In 16 individuals only non-syncytium-inducing (NSI) isolates were detected; syncytium-inducing (SI) isolates were obtained from 3. Studies of transmitter-recipient pairs indicated that both NSI variants and SI variants were transmitted and that SI variants may be suppressed in the recipient. CD4+ T cells remained in the normal range in 15 of 16 individuals with NSI isolates but rapidly declined in all 3 individuals with SI variants, 1 of whom was treated with zidovudine. The most marked increase in CD8+ T cells and activated CD8+ T cells was observed in individuals with the most pronounced clinical signs of acute HIV-1 infection. Activated CD8+ T cells were only transiently elevated in individuals with SI variants, suggesting that an impaired cellular anti-HIV-1 immune response plays a role in the rapid progression to AIDS.


Effect of pili-specific antibodies on the adherence of Haemophilus influenzae type b to human buccal cells.

Different strains of Haemophilus influenzae type b (Hib) produce antigenically distinct pili that mediate adherence to human buccal epithelial cells. This study determined the ability of antibodies specific for the LKP3 pili of Haemophilus influenzae type b to inhibit the adherence of Hib strain Eagan (p+). Antiserum was prepared by immunization of rabbits with pili purified from Hib strain Eagan (p+). The presence of pili-specific antibodies in the immune serum was shown by selective immunoprecipitation of pilin and by electron microscopy of cells labeled with immunogold. Immune serum, affinity-purified antibody, and Fab fragments from immune serum significantly inhibited (P less than .001) adherence of strain Eagan (p+) to human buccal epithelial cells whereas preincubation with preimmune rabbit serum or Fab fragments from preimmune serum had no significant effect on adherence. Dilution of the immune serum, affinity-purified antibodies, or Fab fragments from immune serum resulted in decreased inhibition of Hib adherence. These results indicate that antibodies specific for LKP3 pili can effectively inhibit adherence of Hib strain Eagan (p+) and probably other strains that express this pilus serotype.


Virulent Treponema pallidum activates human vascular endothelial cells.

Perivascular lymphocytic infiltration, fibrin deposition, and endothelial cell abnormalities consistent with cellular activation are prominent histopathologic features of syphilis, a sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum. Because activated endothelial cells play important roles in lymphocyte homing and hemostasis, the ability of virulent T. pallidum to activate cultured human umbilical vein endothelial cells (HUVEC) was investigated. T. pallidum induced the expression of intercellular adhesion molecule-1 (ICAM-1) and procoagulant activity on the surface of HUVEC. Electron microscopy of T. pallidum-stimulated HUVEC revealed extensive networks of fibrin strands not observed in cultures without treponemes. ICAM-1 expression in HUVEC also was promoted by a 47-kDa integral membrane lipoprotein purified from T. pallidum, implicating a role for spirochete membrane lipoproteins in endothelial cell activation. The combined findings are consistent with the pathology of syphilis and provide the first evidence that a pathogenic spirochetal bacterium such as T. pallidum or its constituent integral membrane lipoprotein(s) can activate directly host vascular endothelium.


Immunologic markers of clinical evolution in children recently infected with Leishmania donovani chagasi.

The study attempted to identify immunologic markers for progression of Leishmania donovani chagasi infection to disease in children in an area endemic for visceral leishmaniasis (VL). [3H]thymidine uptake of lymphocytes stimulated with L. donovani chagasi antigen from children with asymptomatic infection (25,286 +/- 11,648) and from children with self-healing subclinical infection (15,511 +/- 4681) was greater (P = .001) than that observed with lymphocytes from children who progressed to classic VL (4811 +/- 2984). The interferon-gamma (IFN-gamma) levels from asymptomatic and subclinically infected children (74 +/- 90 units/ml) were higher (P = .02) than those observed in children who progressed to VL (7 +/- 8 units/ml). Absence of lymphocyte blastogenesis and IFN-gamma production were associated with progression of infection to classic VL. In the presence of these markers, children should be closely followed to identify signs and symptoms that would permit early initiation of therapy.


Antibody to capsular polysaccharides of Streptococcus pneumoniae after vaccination of human immunodeficiency virus-infected subjects with 23-valent pneumococcal vaccine.

The Centers for Disease Control recommends that, because of a greatly increased susceptibility to pneumococcal infection, all persons infected with human immunodeficiency virus (HIV) receive pneumococcal vaccine. Using an ELISA specific for antibody to capsular polysaccharide, a postvaccination antibody was evaluated to five commonly infecting serotypes of Streptococcus pneumoniae. Thirty-nine HIV-infected persons with less than or equal to 500 CD4 cells exhibited significantly fewer responses than did healthy controls; overall, only 46 (24%) of 195 possible responses were positive compared with 45 (75%) of 60 in 12 HIV-infected subjects with greater than 500 CD4 cells and 92 (74%) of 125 in 25 healthy controls (P less than .001). Subjects with less than or equal to 500 CD4 cells responded to a mean of 1.1 antigens versus a mean of 3.8 and 3.7 in those with greater than 500 CD4 cells and controls, respectively (P less than .001). There were no differences between responses in those with less than 200 and those with 200-500 CD4 cells. Within groups stratified by CD4 cell counts, further stratification by clinical status did not reveal significant differences. Since asymptomatic HIV-infected persons with less than 500 CD4 cells show abnormal responses, pneumococcal vaccine should be given when HIV infection is first detected.


Analysis of DNA restriction fragment length polymorphism extends the evidence for breast milk transmission in Streptococcus agalactiae late-onset neonatal infection.

Analysis of restriction fragment length polymorphism (RFLP) of total DNA and of ribosomal DNA (ribotyping) was used to document four cases of Streptococcus agalactiae mother-to-infant transmission potentially associated with ingestion of infected mother's milk. Twenty strains were analyzed. Ten strains were mother-baby pairs, five from the milk of five mothers, four from their neonates with late-onset infection, and one from a colonized neonate. All mothers had early postpartum mastitis. Ten unrelated strains were studied for comparison. In each case, the two strains of each mother-baby pair produced identical RFLP patterns of total DNA. The 10 unrelated strains generated 10 different patterns, one of which, though, was observed in one of the mother-baby pairs. Ribotyping was less discriminative than total DNA RFLP analysis (6 different patterns vs. 13). These data extend the evidence for breast milk transmission in S. agalactiae late-onset neonatal infection.


Disseminated Mycobacterium avium complex infection: clinical identification and epidemiologic trends.

To evaluate the incidence of disseminated Mycobacterium avium complex infection (DMAC) and to define the association between signs and symptoms and development of DMAC in patients with human immunodeficiency virus (HIV) infection, all cases of DMAC at Grady Memorial Hospital Infectious Disease Clinic (Atlanta) between 1985 and 1990 were reviewed, and a prospective study of the association of symptoms with DMAC was done. Between 1985 and 1990, DMAC occurred in 16% of patients with AIDS. Incidence increased from 5.7% in 1985-1988 to 23.3% in 1989-1990 (P less than .001). Median time from AIDS diagnosis to diagnosis of DMAC increased from 4.5 months in 1985-1988 to 8 months in 1989-1990 (P less than .02). In the prospective study, DMAC was seen only in persons with a CD4+ count less than 100 cells/mm3 and was associated with fever (P less than .03), anemia (P less than .001), weight loss (P less than .01), diarrhea (P less than .01), and elevated alkaline phosphatase (P less than .01). It is recommended that all such HIV-infected persons have mycobacterial blood cultures done.


Free radicals from irradiated lyophilized DNA: influence of water of hydration.

Lyophilized DNA equilibrated with water vapour at various relative humidities (0-95% H2O or D2O) was X-irradiated at 77 K and analysed for free radicals by electron paramagnetic resonance (EPR) spectroscopy in the temperature range 77-280 K. Analysis of spectra according to variation in humidity, microwave power and temperature generally yielded a doublet and a triplet spectrum at 77 K. The doublet partially converted into the 5-thymyl radical (TH.). DNA containing deuterated thymine (dTDNA) revealed that the doublet of 'normal' DNA should be composed of two similar doublets, one of which should be assigned to the thymine anion, the other possibly the cytosine anion. The triplet signal was more stable and could be related to the guanine cation or its deprotonated successor. Several other patterns were detected, among them an allyl radical in highly aquated DNA (95% humidity). Other features occurred either predominantly or exclusively in DNA equilibrated above 66% relative humidity and were ascribed to an influence of the secondary structure. Among them were components possibly indicating H- or D-addition to the cytosine base, a reaction also derivable for dTDNA. Quantitative analysis of the total radical yield and the relative TH. concentration revealed that one cause of the dominance of the latter is its thermal stability vs. other species. The total radical concentration increases with target size up to 76% relative humidity, the hydration water forming an integral part of the DNA. At 95% relative humidity OH. radicals are formed in addition to DNA radicals, showing that ice and hydrated DNA have separated into discrete targets.


Level of DNA double-strand break rejoining in Chinese hamster xrs-5 cells is dose-dependent: implications for the mechanism of radiosensitivity.

Rejoining of DNA double-strand breaks (dsb) was measured in a dsb repair-deficient mutant of CHO cells, xrs-5, after exposure to various doses of X-rays in the range between 15 and 50 Gy. For the experiments plateau-phase cultures were employed and dsb assayed by a pulsed field gel electrophoresis assay, the asymmetric field inversion gel electrophoresis (AFIGE). The half-times of dsb rejoining were larger in xrs-5 than in parental CHO cells and increased in both cell lines with increasing dose of radiation. The fraction of dsb remaining unrejoined after 240 min incubation at 37 degrees C was also higher in xrs-5 than in CHO cells, but decreased with decreasing dose of radiation. Although a decrease in the fraction of unrepaired dsb with decreasing dose has also been reported for repair-proficient cell lines, the extent of the phenomenon and its dependence on dose are entirely different in xrs-5 cells. We propose that this decrease in the fraction of unrejoined dsb with decreasing dose of radiation derives from the genetic alterations underlying the increased sensitivity to radiation of xrs-5 cells, and should be considered whenever results at the DNA level are correlated to results at the cell level. It is likely that similar responses will also be observed in other radiation-sensitive mutant cell lines deficient in dsb repair. There was no difference in the induction of dsb per Gy and dalton, as measured with AFIGE, between CHO and xrs-5 cells tested either in the exponential or in the plateau phase of growth.


Chromosome damage induced by restriction endonucleases recognizing thymine-rich DNA sequences in electroporated CHO cells.

The influence of BrdU substitution of DNA in Chinese hamster cells on the frequencies of chromosomal aberrations induced by three restriction endonucleases which recognize thymine-rich sequences in DNA has been studied. The restriction enzymes chosen were Eco RI (recognition site G/AATTC), Sca I (AGT/ACT), and Dra I (TTT/AAA). A restriction enzyme that does not have thymine in the recognition sequence, Hae III (GG/CC), was also tried. These enzymes were introduced into cells by electroporation after two cell cycles of BrdU substitution and the aberration yields compared with that observed in non-substituted cells. Our results seem to indicate that the BrdU-substituted chromatin becomes resistant to the chromosome-breaking activity of the restriction enzymes recognizing thymine-rich DNA sequences. These observations are compared with the patterns of cutting of isolated DNA as shown by agarose gel electrophoresis.


A potential pitfall in the use of electroporation: cellular radiosensitization by pulsed high-voltage electric fields.

Chinese hamster ovary cells have been exposed to high-voltage electric fields causing electroporation (EP) and the interaction between EP and radiation-induced cell lethality investigated. There was a voltage-dependent decrease in plating efficiency, assessed immediately following EP, and cell viability, assessed at 24 h. A linear decrease was seen for both. These decreases were accompanied by a voltage-dependent increase in cell volume, assessed immediately following EP. A good correlation between increases in cell volume and decreases in plating efficiency was seen (r = -0.91). The application of electric fields immediately prior to, or following, irradiation led to a radiosensitization of the cells. This radiosensitization still occurred when a 6 h interval was left between radiation and EP but was lost when cells were irradiated 24 h prior to EP. When cells were irradiated following EP, the radiosensitization was lost with a 1 h interval between the two treatments. These results suggest that, when studying the combined cellular effects of EP of macromolecules and radiation, care should be taken that sufficient time has elapsed between the two modalities to prevent the radiosensitization of cells.


Chromosomal aberrations in human lymphocytes induced in vitro by very low doses of X-rays.

This paper presents results of a collaborative experiment between six laboratories which examined the yields of unstable chromosomal aberrations in human lymphocytes induced in vitro by X-rays over the dose range 0-300 mGy. The work included data points of nominal doses of 0, 3, 5, 6, 10, 20, 30, 50 and 300 mGy. Cells from 24 donors were examined and a total of about 300,000 metaphases were scored. The work was undertaken to determine the limits of sensitivity of the system taking into account variations in scoring data due to inter-donor sample and inter-laboratory effects. Despite the existence of these effects, aberration yields significantly in excess of control values were seen at doses greater than 20 mGy and these were consistent with a linear extrapolation from higher doses. Below 20 mGy the observed dicentric yields were generally lower than background, but not significantly so. Excess acentric aberrations, on the other hand, and centric rings, were higher than the controls but the increase was usually not significant. It is concluded that the statistical uncertainties are such that below 20 mGy this technique cannot distinguish between a linear or a threshold model.


The relative biological effectiveness of 60Co gamma-rays, 55 kVp X-rays, 250 kVp X-rays, and 11 MeV electrons at low doses.

The relative biological effectiveness (RBE) of selected low-LET radiation modalities (55 kVp X-rays, 250 kVp X-rays, 60Co gamma-rays, and 11 MeV electrons) was investigated for survival of two cell lines (V79 and CHO). Detailed measurements were made in the low (0 to 3 Gy) dose range using an image cytometry device to accurately determine the number of cells assayed at each dose point. Data were also collected in the high dose range (0 to 10 Gy) using conventional counting and plating techniques. RBE values (+/- 1 SE) varied from 1.0 +/- 0.07 (V79 cells) and 1.2 +/- 0.05 (CHO cells) at high doses to 1.3 +/- 0.07 (V79) and 1.4 +/- 0.1 (CHO) at low doses for 55 kVp X-rays, from 1.1 +/- 0.05 (V79) and 1.1 +/- 0.04 (CHO) at high doses to 1.1 +/- 0.06 (V79) and 1.2 +/- 0.2 (CHO) at low doses for 250 kVp X-rays, and from 1.1 +/- 0.08 (V79) and 1.0 +/- 0.04 (CHO) at high doses to 1.0 +/- 0.06 (V79) and 0.9 +/- 0.1 (CHO) at low doses for 11 MeV electrons. Only the low and high dose RBEs for 55 kVp X-rays relative to 60Co gamma-rays were significantly different.


Effects of low-dose X-irradiation on mouse-brain aggregation cultures.

Biochemical and morphological differentiation in reaggregating mouse-brain cell cultures after low-dose radiation (0.5 Gy) in vitro was studied. Cells were irradiated on culture day 2, corresponding to embryonic day 15-16, and different glial and neuronal markers were followed through development to postnatal day 40. The shape and size of irradiated aggregates were more irregular and smaller compared with controls. Total amounts of DNA and protein were significantly lower in irradiated aggregates than in controls between days 8 and 20. After 30 days in culture activities of the glial markers glutamine synthetase (GS) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) were lower in X-irradiated aggregates than in controls. However, after 40 days the CNP activity in irradiated aggregates increased to levels above those of the controls. Irradiated and control aggregates did not differ significantly in neuronal marker enzyme activities, i.e. choline acetyltransferase (ChAT), acetylcholine esterase (AChE) and glutamic acid decarboxylase (GAD) measured on a per mg protein basis. On days 20 and 30 the amount of nerve growth factor (NGF) was two-fold higher in irradiated aggregates compared with non-irradiated ones, suggesting that, after irradiation, surviving cells in culture were induced to produce more NGF. After 40 days the amount of NGF in irradiated aggregates had decreased to the level found in the control aggregates.


Increased radiation tolerance of mouse tongue epithelium after local conditioning.

The effect of local stimulation on mitotic activity and radiation tolerance was studied in mouse tongue mucosa. Silver nitrate solution (0.5-20%) was used for local conditioning. The most effective protocol comprised three daily treatments (days 0-2), yielding a delayed increase in 24 h mitotic counts by about 30% on days 5-7. The stimulating effect was independent of silver nitrate concentration. Sham treatment with saline or anaesthesia alone clearly depressed mitotic activity on days 2-4 without any subsequent overshoot. Radiation treatment was initiated on day 5 after three daily treatments with 3% silver nitrate solution. A top-up technique was employed, consisting of fractionated irradiation (300 kV X-rays) of the whole snout, followed by graded local test doses (25 kV X-rays) to induce denudation in a confined area of the inferior tongue surface. Silver nitrate conditioning did not alter the radiosensitivity of the epithelium to single local doses, but shortened the latency to denudation from 11 to 8 days. In contrast, a clear increase in tolerance to fractionated irradiation, delivering 5 x 2.5, 5 x 3.5, 5 x 4.5 Gy or 3 x 5.2 Gy in 7 days, was observed, equivalent to about four, two, one and two extra dose fractions. This approach may be a suitable way to increase radiation tolerance of oral mucosa in clinical radiotherapy.


The influence of lumenal pH on the severity of acute radiation enteritis.

The severity of acute radiation injury to small bowel mucosa was studied as a function of lumenal pH at the time of irradiation. Rats were anaesthetized and 15 cm of mid small bowel was exteriorized with the rest of the animal and intestine shielded in a lead box. The target intestine was segmented into three portions, each of which was filled with Tris buffer, adjusted to pH 5, 7 and 9 respectively. Twenty minutes later the exteriorized bowel was given 1100 cGy X-irradiation. Rats were sacrificed 4, 5 and 6 days later and the intestine histologically assessed for injury. The indices of injury were surviving crypt numbers, mucosal height and mucus-containing goblet cells. To verify that pH was the critical variable an identical study was done employing three buffers chemically different from Tris: MES (pH 5.5), ADA (pH 7), and CAPSO (pH 9). These animals were sacrificed 5 days postirradiation. For all three parameters, and on each day postirradiation, the pH 9 segment showed less damage than did the segments with lumenal pH 7 or 5. We conclude that lumenal pH at the time of irradiation plays a significant role in the severity of acute mucosal injury. Alkaline pH is relatively protective.


Antimutagenic effects of radioprotector WR-2721 against fission-spectrum neurons and 60Co gamma-rays in mice.

The antimutagenic effects of the radiation protective agent, S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721), were studied against fission-spectrum-neutron- and 60Co-gamma-ray-induced mutagenesis in mice. Mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus was measured 56 days following whole-body irradiation with JANUS neutrons (single doses, 50-150 cGy) or 60Co photons (single doses, 250-750 cGy). Splenic T lymphocytes from B6CF1 mice were grown in round-bottomed 96-microwell culture plates with or without the selective agent 6-thioguanine (6-TG). The mutant frequency, as a result of exposure to neutrons or 60Co photons, increased 100-fold with dose. Doses of 150 cGy neutrons and 750 cGy 60Co photons were equally mutagenic. When animals were injected with WR-2721 at a dose of 400 mg/kg body weight, i.p., 30 min before whole-body irradiation with JANUS neutrons or 60Co photons, mutant frequencies were significantly reduced at all radiation doses (i.e. protection factors of 1.4 and 2.4, respectively). Thus, the aminothiols are effective antimutagens. A novel clinical application of these compounds could be in their use to protect against radiation- and/or chemotherapy-induced genotoxic damage to normal cells.


Alpha-irradiation of haemopoietic tissue in pre- and postnatal mice: 2. Effects of mid-term contamination with 239Pu in utero.

The distribution of 239Pu in various tissues of foetal and postnatal offspring of pregnant mice, injected i.v. at 13 days gestation with 30 kBq 239Pu/kg (in some cases with 10 or 100 kBq/kg), together with the numbers of haemopoietic progenitors in the bone marrow, spleen and liver, were measured through to 1 year post-partum. The quality of the haemopoietic microenvironment in these mice was also measured using the renal-capsule implant method. The largest radiation dose received by any haemopoietic organ was that in the liver, amounting to 10-14 mGy, as reported previously. In spite of normal numbers of haemopoietic spleen colony-forming cells (CFC-S) in the liver and seeding, at birth, into the bone marrow where the level of plutonium was minimal, a long-term deficit in their number rapidly developed. The development of the stromal microenvironment, however, was also deficient, suggesting that the dose of alpha-irradiation to the foetal liver was sufficient to cause sublethal damage in those cells destined to become the precursors of the supportive haemopoietic microenvironment in bone marrow and spleen. The results of this study suggest that although the placenta affords significant shielding to the tissues of the developing foetus from maternal contamination, the long-term effects on haemopoiesis are comparable to those in mice contaminated as adults. This further implies that the developing haemopoietic tissues are exquisitely sensitive to 239Pu contamination.


Expression of oestrogen receptor and transforming growth factor-alpha in MCF-7 cells after exposure to fractionated irradiation.

The expression of critical growth controlling genes was studied in MCF-7 cells after exposure to cumulative radiation doses of 20 and 60 Gy yielding cell lines called MCF-IR-1 and MCF-IR-3, respectively. The irradiated cell lines exhibited increased plating efficiencies but no differences in growth rates. MCF-IR-1/-IR-3 cells showed a reduced oestrogen responsiveness as indicated by their diminished response to tamoxifen-induced growth arrest and 17 beta-oestradiol (E2)-induced growth stimulation. The reduced expression of oestrogen receptor (ER) was determined by quantitative immune peroxidase staining of single cells and by total cellular E2 binding. There was also a radiation dose-dependent increase in the radiosensitivity of MCF-IR-3 cells as determined by the radiobiological parameters alpha, beta, and D (mean inactivation dose). Using RNA protection assays the irradiated cell lines produced steady-state ER mRNA at reduced levels while the levels of TGF-alpha were unchanged in MCF-IR-1 cells but increased 2.8-fold in MCF-IR-3 cells. A similar pattern was seen for TGF-alpha protein. While the current analyses cannot differentiate between radiation-induced altered gene expression or cell selection the results demonstrate that reduced ER expression and increased TGF-alpha expression are associated with the survival of MCF-7 cells after fractionated irradiation in vitro. In contrast, the MCF-IR cells were found to be more radiosensitive in acute survival experiments.