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The aim of this study is to isolate and characterize cDNA clones and the genes that encode mouse RPE retinal G protein-coupled receptor (RGR) and to analyze expression of the RGR gene in the developing mouse retina. The conserved amino acid sequences of RGR from various mammals can be compared to the amino acid sequence motif of G protein-coupled receptors.Mouse RGR cDNA and gene clones were isolated from a retina cDNA library and 129SV genomic DNA library, respectively. The expression of RGR in the developing C57BL/6J mouse retina was analyzed by immunohistochemical staining with a polyclonal antipeptide antibody.The deduced amino acid sequence of mouse RGR is 78% and 81% identical to that of bovine and human RGR, respectively. The mouse RGR gene is split into seven exons and extends about 11 kb. Two predominant mRNA transcripts, 1.9 and 1.7 kb in length, and a third, relatively faint, 5.5-kb transcript were detected in mouse eye by hybridization to a RGR cDNA probe. Frozen sections of C57BL/6J mouse retina at various stages of development were incubated with a mouse RGR antipeptide antibody. RGR immunoreactivity was first seen at postnatal day 2 (P2) in centrally located RPE cells. From day P6 to P12, there was an increase in the number and intensity of immunoreactive RPE cells in the central and mid-peripheral regions of the retina, while the most peripheral RPE cells were still negative. By day P16, the length of the RPE monolayer was immunoreactive, and staining of the central RPE cells was markedly more intense than at younger ages.Mouse and human RGR are highly conserved. A gradient of RGR expression in RPE extends from the central to the peripheral retina during development. In reference to the appearance of melanin-positive differentiated RPE cells, the induction of RGR expression is a relatively late event in the maturation of the retina.

Ionizing radiation (IR) results in apoptosis in a number of actively proliferating or immature cell types. The effect of IR on rat dorsal root ganglion (DRG) neurons was examined in dissociated cell cultures. After exposure to IR, embryonic DRG neurons, established in cell culture for six days, underwent cell death in a manner that was dose-dependent, requiring a minimum of 8 to 16 Gy. Twenty-five per cent cell loss occurred in embryonic day 15 (E-15) neurons, grown in cell culture for 6 days ('immature'), and then treated with 24 Gy IR. In contrast, only 2% cell loss occurred in E-15 neurons maintained in culture for 21 days ('mature') and then treated with 24 Gy IR. Staining with a fluorescent DNA-binding dye demonstrated clumping of the nuclear chromatin typical of apoptosis. Initiation of the apoptosis occurred within 24 h after exposure to IR. Apoptosis was prevented by inhibition of protein synthesis with cycloheximide. Apoptosis induced by IR occurred more frequently in immature than in mature neurons. Immature DRG neurons have a lower concentration of intracellular calcium ([Ca2+]i) than mature neurons. Elevation of [Ca2+]i by exposure to a high extracellular potassium ion concentration (35 microM) depolarizes the cell membrane with a resultant influx of calcium ions. The activation of programmed cell death after nerve growth factor (NGF) withdrawal is inversely correlated with [Ca2+]i in immature DRG neurons. When treated with high extracellular potassium, these immature neurons were resistant to IR exposure in a manner similar to that observed in mature neurons. These data suggest that [Ca2+]i modulates the apoptotic response of neurons after exposure to IR in a similar manner to that proposed by the "Ca2+ setpoint hypothesis" for control of NGF withdrawal-induced apoptosis.

Several approaches, including immunoquantification of individual enzymes, profiling of substrate activities by immunoinhibition using highly specific polyclonal and monoclonal antibodies, and the estimation of corresponding mRNAs with nucleic acid probes, have been used to investigate the ontogeny of cytochromes P450 in livers of rodents and man. CYP1A1 is expressed very early in development in rodents, whereas most other enzymes either appear at or near birth (CYP2B, CYP2C23, and CYP3A) or between 2 and 4 weeks following birth (CYP2A, CYP2C6, CYP2C7, CYP2C11, CYP2C12, and CYP4A10). The constitutive expression of enzymes is subject to regulation by various transcriptional nuclear and/or hormonal factors (CYP2B and CYP2C) or in a sex-dependent manner (CYP2A, CYP2C11, CYP2C12, CYP3A, and CYP4A10). The enhanced sensitivity and specificity of immunocytochemical and in situ hybridisation studies have revealed differences, with age and xenobiotic treatment, in the intercellular expression of certain P450 enzymes of the liver. For example, in rats, the expression of CYP1A1 and 1A2 is differentially regulated at the level of the individual cell from as early as 24 hours before birth. The human foetal liver relative to rodents has a substantial level of CYP3A and also has the capacity to metabolise a greater repertoire of substrates. Evidence to date suggests that P450 enzymes in man are regulated in a manner similar to that in other animals. The balance between different individual enzymes of cytochrome P450 in foetuses and/or neonates is subject to modulation by xenobiotics, the consequences of which may lead to toxicologically compromised livers with respect to metabolic handling of certain substrates.

Mouse retinal photoreceptor cell generation and morphogenesis take place in a well-characterized temporal sequence. Both rod and cone photoreceptor differentiation and synaptogenesis occur postnatally, but the relative timing of these events has been difficult to document due to the paucity of cell-specific markers. We have found that antibodies to neuron-specific enolase (NSE) preferentially label a subpopulation of photoreceptors in the outer nuclear layer (ONL) of the mouse retina in addition to labeling ganglion, amacrine, bipolar, and horizontal cells within the inner layers of the retina. The appearance of NSE immunoreactivity in the different classes of retinal neurons during development showed a close temporal relationship to the onset of expression of the synaptic vesicle-associated protein SV2 and clearly preceded the sequential development of synaptic connections in both inner and outer synaptic layers. The NSE-immunoreactive photoreceptors were identified as cones by dual labeling of their inner segments with the lectin peanut agglutinin or by colabeling with antisera to cone photopigments. Axonal extensions of NSE-labeled cone cells were shown to interact with those of differentiating horizontal cells as early as postnatal day 3 (P3). Colocalization of NSE with SV2 indicated that cone cells began to make synaptic contacts with horizontal cell processes several days prior to the development of rod synaptic terminals. Between P4 and P11, cone photoreceptor cell nuclei were observed to be scattered at various levels throughout the ONL and thus appeared to have become displaced from their previous position directly beneath the outer limiting membrane (OLM). By P12, the cone nuclei had migrated sclerad once again and were now observed to be neatly aligned adjacent to the OLM. In the rd mouse mutant, this migratory process was delayed, so that, at P12, positioning of the cone cell nuclei within the ONL was still quite irregular. Thus, we have identified a late migratory phase for cone photoreceptors during the second week after birth that correlates with the timing of maturation of the rod synaptic terminals just prior to eye opening. The types of cues used by maturing cone cells for their eventual sclerad location remain to be elucidated.

The associations among timing of maternal-fetal human immunodeficiency virus (HIV) transmission, infant age at first positive HIV culture, and CD8+ lymphocyte activation were examined for 74 perinatally infected infants. Nineteen of the infected infants had positive HIV cultures ator =7 days of life, and 55 had negative HIV cultures ator =7 days but were positive later. Of the infants with early positive HIV-1 cultures, 15 of the 17 tested with DNA polymerase chain reaction methods had concordant results. The percentage of CD8+ and HLA-DR+ lymphocytes (CD8+DR+%) during the first week of life was significantly higher in infants with early compared with late positive cultures (median CD8+DR+% of 5.0% versus 2.0%, p = 0.0006). The CD8+DR+% was similar between uninfected infants and infants with late positive cultures during the first week of life (median 2%) but increased in infants with late positive cultures to 6% by 1 month. The CD4+% during the first 6 months of life was not different between infants with early or with late positive cultures, but infants with the highest CD8+DR+% ator =7 days of life had significantly lower CD4+% ator =7 days and at 1, 2, and 4 months of age. These data show that early CD8+ lymphocyte activation is associated with early positive HIV cultures and lower CD4+ percentages during early infancy and are consistent with the hypothesis that early positive cultures positivity may indicate in utero HIV infection.

A retroviral marker was used to label daughter cells arising from individual neuroblasts in the rd mouse retina, in order to investigate the hypothesis that a clonal relationship exists among degenerating photoreceptor cells.On the day of birth, a single injection of retrovirus with a lac Z (beta-galactosidase) reporter construct was injected into the retina in the vicinity of the subretinal space. Descendants of single neuroblasts were identified histochemically by examining the retinas at P14 (postnatal day 14). Light and electron microscopic studies were used to identify the retrovirally-induced marker beta-galactosidase using Bluo-gal dye. Double-labeling of degenerating cone cells was accomplished by taking 100 microns vibratome sections of retrovirally-injected eyes and using either FITC-PNA or HRP-PNA to visualize clusters of degenerating cones as well as Bluo-gal labeled clones of photoreceptor cells on the same tissue section.A relatively large number of clones of primarily photoreceptor cells were observed in the peripheral retinas of both normal and rd mice. In a few cases in the rd, photoreceptor cells in a given clone consisted of both PNA- and Bluo-gal-labeled cells as well as of only Bluo-gal-labeled cells.These results suggest that during the period of intense cell death in the rd retina, a single dying photoreceptor cell can be surrounded by photoreceptors (either rods or cones) from the same clone that appear morphologically normal without evidence of degeneration.

Selective degeneration of rod photoreceptor cells in the retinal degenerative (rd) mouse prior to their complete maturation is thought to result from elevated cyclic guanosine monophosphate (cGMP) levels owing to the inherited defect in cGMP-phosphodiesterase. To investigate potential signaling pathways which might lead to apoptotic death of photoreceptors in the rd retina, the expression of immediate-early genes (IEG) of the activating protein-1 transcription factor (AP-1) family was examined. Increasing numbers of apoptotic photoreceptor nuclei were observed in the outer nuclear layer of the rd mouse beginning at postnatal day (P) 10. The peak incidence of apoptotic cells was observed at P13; by P16, almost the entire population of photoreceptors had been lost. Although c-Fos-like immunoreactivity was absent in photoreceptors of normal retinas, we observed that commencing at around P10, increasing numbers of rod photoreceptors in the rd retina exhibited nuclear staining for c-Fos protein. While no change in the distribution patterns of other members of the AP-1 family (c-Jun, JunB, and JunD) was observed in photoreceptors, Müller cell nuclei were transiently immunoreactive for c-Jun on P11. The incidence of c-Fos-positive photoreceptors peaked sharply at P12, 1 day earlier than the peak in apoptosis. Furthermore, the population of c-Fos-positive photoreceptors was distinct from apoptotic photoreceptors exhibiting chromatin condensation. The aberrant expression of c-Fos protein in rod photoreceptors immediately prior to their death in the rd mouse raises the possibility that c-Fos may be directly or indirectly involved in triggering the apoptotic cascade. Furthermore, the additional finding of c-Jun induction in Müller glia suggests that the IEG response to photoreceptor degeneration involves both intra- and intercellular signal transduction pathways.

Immature rat facial motoneurons are very sensitive to injury with nearly 80% dying during the first week after axotomy. This motoneuron death is apoptotic, similar to that induced in neurons after tropic factor withdrawal. The diphenylpiperazines, flunarizine and cinnarizine, protect dorsal root ganglion neurons from death after withdrawal of trophic support, i.e., nerve growth factor withdrawal, in vitro. Similarly, the monoamine oxidase inhibitor, deprenyl, promotes survival of facial motoneurons after axotomy. These pharmacological agents were assessed both alone and in combination for their ability to prevent death in non-nerve growth factor dependent CNS motoneurons after facial nerve axotomy in newborn rats. Long-term experiments were done with the diphenylpiperazines to evaluate potential enhancement of regeneration. Facial nerve transection resulted in 78% neuronal loss in the injured compared with the contralateral, uninjured nucleus. Systemic administration of diphenylpiperazines for 1 week after facial nerve transection doubled the number of surviving motoneurons from 23% to 47%. Similar results were obtained with deprenyl. Combinations of diphenylpiperazines and deprenyl provide a similar degree of neuronal protection 1 week after injury as that obtained by either agent alone. We assessed the ability of diphenylpiperazines to protect facial motoneurons from death over a prolonged period and enhance subsequent regeneration. Motor neuron counts in rats treated with diphenylpiperazines for 1 month after injury and assessed 2 months later demonstrated long-term enhancement of neuronal protection with an increase of 45% in the number of horseradish peroxidase-labelled motoneurons. The diphenylpiperazines group had approximately 80% more regenerated myelinated axons in the distal facial nerve than the control group. Thus, diphenylpiperazine treatment during the first month after injury provides long-term protection of non-nerve growth factor dependent CNS motoneurons with subsequent potentiation of long-term facial nerve regeneration.

MeIQx, a potent bacterial mutagen formed when meat is cooked, requires metabolic activation to exert its genotoxicity, a reaction catalysed primarily by CYP1A2 in adult mammals. Little is known about mammalian developmental changes in the mutagneic activation of compounds such as MeIQx. In rabbits we have shown previously that expression of CYP1A2 increases with age and is inducible by 3-methylcholanthrene (MC) from as early as 4 days pre-parturition. We have therefore investigated the effect of age on rabbit liver activation of MeIQx (assessed in an Ames test using Salmonella typhimurium TA98) before and after treatment of the animals with MC. MeIQx activation could not be detected using hepatic microsomal fractions from rabbits of

From February 1989 to December 1993, 139 patients with a variety of brain lesions were treated with stereotactic external beam irradiation using a 6MV linear accelerator. The largest group consisted of patients with recurrent brain metastases (n = 46). Twenty seven patients had malignant gliomas, most of which were recurrent. Several benign conditions were treated, including arteriovenous malformations (n = 27), acoustic neuromas (n = 9), meningiomas (n = 7), and pituitary adenomas (n = 2). Durable responses were seen in the majority of patients with transient, mild, side effects. This experience suggests that stereotactic external beam irradiation is a safe, reliable, and effective method for non-invasive treatment of selected patients with small, localized brain lesions.

2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are amongst the most abundant of the heterocyclic aromatic amines formed during the cooking of beef. Both compounds are genotoxic and carcinogenic in rodents. These effects are manifested only after activation of the amines by P450. Human liver is very active at converting these amines to mutagenic products. Studies in vitro have established that, for both amines, mutagenicity with human liver microsomes is entirely via the N-hydroxylamine, essentially the only oxidation product of either amine. Both N-hydroxylation and mutagenicity of the amines can be almost completely inhibited by furafylline, a potent and highly selective inhibitor of CYP1A2 in man. These data, together with the work of others, show that the N-hydroxylation and hence the mutagenicity of both MeIQx and PhIP in man is catalyzed almost exclusively by CYP1A2. Liver from cynomolgus monkeys, unlike that from humans and marmosets, is very poor at activating MeIQx or PhIP to mutagenic products. Studies with anti-peptide antibodies of defined specificity revealed that this is due to the absence of CYP1A2, suggesting that this species should not be used to assess the possible risk posed to man by these amines. The marmoset would be a better species for this purpose.

We investigated the expression, distribution, and inducibility of 3-methylcholanthrene (MC)-inducible P450 enzymes, CYP1A1 and 1A2, in livers of rabbits at different stages of development, ranging from 4 days before birth (-4 days of age) to adulthood. These enzymes were identified by immunoblotting and immunocytochemistry and quantified by dot-blotting, utilizing previously characterized monoclonal antibodies, 107 and 3/4/2, specific for CYP1A2 and both CYP1A1 and 1A2, respectively, and a polyclonal antibody that recognizes both enzymes. Expression of CYP1A2 is always greater than that of CYP1A1 in livers of untreated rabbits, regardless of age. Moreover, immunocytochemistry showed that CYP1A1 is evenly distributed throughout the liver at all ages, whereas CYP1A2 is highly localized to only a few scattered cells at 1 day before birth. More hepatocytes express this enzyme perinatally. By 6 days of age, expression of CYP1A2 is confined to a narrow band of centrilobular cells, but with increasing age the enzyme is expressed in more hepatocytes until weaning, when all hepatocytes are positive. Although CYP1A1 is induced by MC treatment at most ages, there is no change in its distribution. In contrast, induction of CYP1A2 was shown immunocytochemically to occur in only a limited number of hepatocytes in fetal rabbits. There is a progressive increase with age in the number of hepatocytes that are inducible for CYP1A2. The greatest fold-induction of hepatic CYP1A2 by MC in the rabbit is a 9-11 days of age, when, for MC-treated rabbits, CYP1A2 represents > 60% of the total P450 pool. The modulation of enzyme expression caused by MC treatment of fetuses/neonates leads to developmentally advanced livers with respect to P450 and could have a significant impact on the fetal and neonatal toxicity of some foreign compounds. These data demonstrate, for the first time, that the ontogenetic expression and localization of CYP1A1 and 1A2 within the liver are differentially regulated at the level of the individual cell.

In this study, 29 formalin-fixed, paraffin-embedded astrocytic tumors were analyzed immunocytochemically with the antikeratin monoclonal antibodies Mak-6 and Cam 5.2 and a polyclonal antibody against glial fibrillary acidic protein (GFAP). Immunoreactivity for Mak-6 was present in 29 cases (100%) including six well-differentiated astrocytomas, nine anaplastic astrocytomas, and 14 glioblastomas multiforme. Cam 5.2 immunoreactivity was focally present in one case of GBM (4%) but was absent in the remaining 28 cases. All cases were immunoreactive with an antibody against GFAP. Cytokeratin (CK) expression was examined in extracts of four separate well-characterized astrocytoma cell lines by Western blotting with the monoclonal antibodies Mak-6, Cam 5.2, and anti-CK 18 and by Northern analysis using a cDNA probe for the human CK 18 gene. The Western blots revealed the presence of immunoreactive bands corresponding to CK numbers 14/15, 16, and 18 in extracts from all four cell lines and additional bands corresponding to CK 8 in 3/4 lines and CK 19 in 1/4 lines. Northern analysis detected CK 18 mRNA in extracts from 2/4 astrocytic cell lines. These findings demonstrate that CK immunoreactivity is frequent in astrocytic tumors and confirm through the molecular and biochemical analysis of CK 18 gene expression and of keratin intermediate filament proteins that the basis for CK immunoreactivity in astrocytic tumors is bona fide CK expression, not cross-reactivity with other antigens or artifact. The demonstration of CK expression by astrocytic neoplasms has important implications for pathologists involved in the diagnosis of poorly differentiated tumors.

To improve standardization of a folate bioassay, folate-depleted rats were repleted with a folate-free amino acid-based diet supplemented with 29 levels of folic acid. Growth was the main response variable and body tissue folate concentrations were also assessed. Because a positive correlation was observed between low levels of dietary folic acid and growth and little or no correlation was observed between high levels and growth, six regression models with a steep slope for low levels and a shallow or zero slope for high levels of dietary folic acid were evaluated. The model referred to as the "two-phase regression" or "change-point" model best described the relationship. Depleted rats needed 674 +/- 71 nmol folic acid/kg diet to reach their full growth potential. This value is biologically sensible, and this regression model is well established in the statistical literature. The change-point model is highly recommended to characterize the growth response, because growth is a functional response and, in the range of 226 to 680 nmol folic acid/kg, this response is linear, which is an additional advantage. Linear responses are easier to interpret because of complicated issues of interpretation and confidence intervals with nonlinearities. Linear regressions described serum and liver folate responses, whereas exponentials described whole blood and carcass folate responses. Depleted rats needed 5920 and 5780 nmol folic acid/kg diet to maximize their whole blood and carcass folates, respectively.

Since the discovery of nerve growth factor (NGF) nearly 40 years ago, extensive studies have elaborated the trophic relationship between a neuron and its end organ. Much evidence has accumulated that depicts the crucial role of neurotrophic factors in determining neuronal survival both during development and after injury. The molecular events by which trophic factors determine whether an individual neuron lives or dies are poorly understood. The function of active protein synthesis as a prerequisite for neuronal death has led to the hypothesis that all neurons contain a mechanism to self-destruct or a phenomenon of programmed cell death. The loss of trophic support may, therefore, lead to the initiation of this so called "suicide" program within individual cells. Recently, multiple other trophic factors have been discovered. At least two of these factors, brain-derived neurotrophic factor and neurotrophin-3, appear related to members of the same gene family as the prototypic neurotrophic factor, nerve growth factor. Other metabolic factors, such as intracellular calcium, may be instrumental in determining the dependence of individual neurons on specific trophic support. A "set-point" hypothesis has been described concerning the relationship between intracellular calcium and the NGF dependency of embryonic neurons in cell culture. Changes in calcium metabolism may be an important determinant in altering trophic dependence and naturally occurring cell death. Neurotrophic factors also influence the reaction of the neuron to axotomy and subsequent axonal regeneration. Exogenously supplied trophic factors may prevent neuronal loss after axotomy when supplied at the site of injury. Both NGF and FGF have been shown to enhance early regeneration across gaps within silicone chambers.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies that involve subjects who are HIV infected as well as chemically dependent present special challenges to the study team. HIV infection is now so common in many parts of the country and infection can potentially influence so many commonly used endpoints for perinatal chemical dependence studies that HIV must be considered in these studies. Some of the factors that need to be taken into account in the design of studies include the staging of HIV illness, the consequences of HIV-induced CNS disease, the effects of HIV treatment, and the biases in subject selection that arise due to inaccurate information about risk factors and imbalance in sources of subjects and controls. In addition, the study team needs to remember the community and staff fear and bias that are often encountered in the care of HIV-infected patients. Fruitful sources of subjects for perinatal studies include prenatal clinics and delivery rooms, chemical dependence programs, and the network of community social agencies. Retention of this challenging group is strongly aided by the establishment of a seamless system of services that includes all aspects of general medical care, HIV specialty care, access to treatment protocols, and psychosocial care.

The signaling pathways which lead to the wave of photoreceptor cell death in the rd mouse during the second week after birth have yet to be defined. The photoreceptors of these mice develop normally until they begin to elaborate phototransducing outer segments at around postnatal day (P)8. At this point, their differentiation slows as intracellular levels of cGMP rise due to the inherited mutation in the phosphodiesterase-β subunit.We have used an immunocytochemical approach to investigate potential signaling pathways leading to photoreceptor cell apoptosis in this mutant. The earliest change detected in the photoreceptor layer was an induction of CREB immunoreactivity in photoreceptor nuclei, commencing at P8. Although CREB is generally thought to be constitutively expressed and its function controlled primarily by its phosphorylation, we find that immature photoreceptors of normal mice are immunonegative for CREB until the time of eye opening and the initiation of phototransduction at around P14.

An exogenous fibronectin-laminin (FN-LAM) solution was added into silicone chambers to determine the effects on peripheral nerve regeneration across 18-mm long gaps. The sciatic nerves of adult rats were sutured into silicone chambers 20 mm in length, creating an 18-mm gap between the proximal and distal nerve stumps. The chambers were filled with either a mixture of fibronectin and laminin (500 micrograms/ml each) or a solution of cytochrome C (1 mg/ml) as the control. After six weeks, the animals were killed and the chambers were examined for regeneration. Seventy percent of the animals from the FN-LAM group demonstrated regeneration across the 18-mm gaps, compared to only 30% in the control group. The combination of FN-LAM significantly increased the number of axons that grew into the distal end of the chamber (FN-LAM, 1325 +/- 522; cytochrome C, 153 +/- 104; p = 0.03). Examination of the distal tributaries of the sciatic nerve revealed axons only in the FN-LAM group; none were found in the control group. Quantitative analysis of neurons, retrogradely labeled with horseradish peroxidase via injection of the sciatic nerve distal to the regenerated segment, revealed a greater number of sensory and motor neurons in the FN-LAM group compared to the control group. Morphometric studies revealed that the mean area of the regenerated segment in the FN-LAM group was 37% larger than the controls, and ultrastructural analysis demonstrated a more mature regenerated nerve. This is the first in vivo demonstration that this combination of fibronectin and laminin significantly enhances the regeneration of myelinated axons across a long nerve gap in the rat sciatic nerve.

In purified preparations of human erythrocyte GTP-binding proteins, we have identified a new substrate for pertussis toxin, which has an apparent molecular mass of 43 kDa by silver and Coomassie Blue staining. Pertussis toxin-catalyzed ADP-ribosylation of the 43-kDa protein is inhibited by Mg2+ ion and this inhibition is relieved by the co-addition of micromolar amounts of guanine nucleotides. GTP affects the ADP-ribosylation with a K value of 0.8 microM. Addition of a 10-fold molar excess of purified beta gamma subunits (Mr = 35,000 beta; and Mr = 7,000 gamma) of other GTP-binding proteins results in a significant decrease in the pertussis toxin-mediated ADP-ribosylation of the 43-kDa protein. Treatment of the GTP-binding proteins with guanosine 5'-O-(thiotriphosphate) and 50 mM MgCl2 resulted in shifting of the 43-kDa protein from 4 S to 2 S on sucrose density gradients. Immunoblotting analysis of the 43-kDa protein with the antiserum A-569, raised against a peptide whose sequence is found in the alpha subunits of all of the known GTP-binding, signal-transducing proteins (Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 265-259) showed that the 43-kDa protein is specifically recognized by the common peptide antiserum. A pertussis toxin substrate of similar molecular weight was observed in human erythrocyte membranes, bovine brain membranes, membranes made from the pituitary cell line GH4C1, in partially purified GTP-binding protein preparations of rat liver, and in human neutrophil membranes. Treatment of neutrophils with pertussis toxin prior to preparation of the membranes resulted in abolishment of the radiolabeling of this protein. From these data, we conclude that we have found a new pertussis toxin substrate that is a likely GTP-binding protein.

DEFICIENCY of the purine enzyme nucleoside phosphorylase is associated with a severe T-lymphocyte immune deficiency.1 2 3 4 5 6 The abnormality in the immune system results from the accumulation of...

The hepatic glucagon receptor was covalently labeled with [125I-Try10]monoiodoglucagon [( 125I]MIG) by use of the heterobifunctional cross-linker hydroxysuccinimidyl p-azidobenzoate. Labeling of the Mr = 63,000 peptide was sensitive to glucagon and GTP at concentrations at which they affect [125I]MIG binding to the receptor. The labeled receptor was solubilized with Lubrol-PX, and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are: S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f0 = 1.8, and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees C for 15 min prior to the addition of [125I]MIG permitted us to identify the high molecular weight form (Mr = approximately 113,000) of the receptor by direct sodium dodecyl sulfate-gel electrophoretic analysis. The Mr = 63,000 peptide can be adsorbed to wheat germ lectin-Sepharose. The glycoprotein nature of the receptor has been utilized to develop an assay for the detergent-solubilized receptor that uses wheat germ lectin-Sepharose as a solid matrix to adsorb the [125I] MIG-receptor complex. The free hormone remains in the liquid phase and is removed in the supernatant after low speed centrifugation. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) solubilizes receptors with retention of [125I]MIG binding activity. [125I]MIG binding to the CHAPS-solubilized receptor is specifically affected by unlabeled glucagon. Interaction of [125I]MIG with the soluble receptor is insensitive to the presence of GTP. IC50 for glucagon using the soluble receptor was 33-70 nM, irrespective of the presence or absence of GTP, while when the membrane-bound receptor was used, the IC50 in the absence of GTP was 2-4 nM and in the presence of GTP was 35-80 nM. These data allow us to conclude that the hepatic glucagon receptor in the membrane and in the nondenaturing detergent solution is a dimer of the Mr = 63,000 hormone-binding subunit and a glycoprotein. The soluble receptor does not display any functional interaction with the stimulatory regulator.

Treatment of MDCK cells with glucagon results in decreases in glucagon, NaF and prostaglandin E1-stimulated adenylyl cyclase activities, indicating the occurrence of a heterologous desensitization process. The extent of desensitization was time and glucagon concentration dependent. Maximal desensitization (30-50% decrease in stimulation by various effectors) was obtained by 4 h at 100 nM glucagon. Glucagon also induced homologous desensitization since after treatment, the Kact of glucagon was specifically increased. Treatment of cells with 10 microM 8-bromoadenosine 3':5'-monophosphate or 10 microM forskolin resulted in decreased hormonal (glucagon and prostaglandin E1) stimulation without any decrease in the stimulation by nonhormonal effectors (NaF, forskolin, and guanyl-5'-yl imidodiphosphate). The stimulatory regulator (Ns) of the adenylyl cyclase system was analyzed after desensitization with glucagon and no measurable changes in the apparent levels of the alpha s subunits of Ns or the activity of Ns as assessed by reconstitution of the cyc- S49 cell membrane adenylyl cyclase were detected. Levels of the alpha i subunit of the inhibitory regulator (Ni) were monitored by labeling with [32P]NAD and pertussis toxin. Membranes of glucagon-treated cells showed a 2-fold increase in the amount of alpha i labeled. Addition of pure Ns to glucagon-treated MDCK cell membranes restored full stimulation by NaF but did not restore stimulation by prostaglandin E1 or glucagon. It is concluded that glucagon induces heterologous and homologous desensitization of the MDCK cell adenylyl cyclase. The locus of the heterologous desensitization is at the level of the regulatory components. Decreased stimulation is thought to occur due to either an increase in the levels of Ni or due to altered interactions between the subunits of Ni.

The effect of the glucagon receptor on the activation of the stimulatory GTP-binding protein of adenylyl cyclase (Gs) in the native rat liver membrane environment was studied. The activated state of Gs was assessed by its ability to reconstitute the cyc- S49 cell membrane adenylyl cyclase. The Gs protein was activated by saturating concentrations of guanosine 5'-thiotriphosphate (GTP gamma S) or guanyl-5'-yl imidodiphosphate in a hormone-dependent manner at 0.4 mM Mg2+ in native membranes or in membranes that had been treated with 1 mM N-ethylmaleimide to eliminate the catalytic activity of adenylyl cyclase. At 50 mM Mg2+, Gs was fully activated by GTP gamma S in the absence of hormone. The unactivated Gs protein migrates around 4 S, whereas activated Gs migrates around 2 S on sucrose density gradients. When pure Gs is analyzed on sucrose density gradients, it is found that the unactivated protein migrates at 4.1 S. Gs was activated by saturating concentrations of GTP gamma S and Mg2+, and the alpha subunit of Gs was chromatographically purified. The resolved alpha subunit of Gs that is capable of stimulating the cyc- adenylyl cyclase migrates at 2.1 S. From these data, we conclude that activation of Gs results in the dissociation of this protein in the membrane environment and that the hormone-occupied receptor promotes this dissociation process under conditions where Mg2+ ions are limiting.

The effect of prolonged exposure of S49 murine lymphoma cells to (−) isoprenaline on the levels of β-adrenergic receptors and the stimulatory regulatory component of adenylyl cyclase (Gs) was investigated. Exposure of wild-type S49 cells (WT cells) to isoprenaline for 16 h resulted in an 84% decrease in β-adrenergic receptors and a 78% decrease in catecholamine-stimulated adenylyl cyclase activity. The desensitization of adenylyl cyclase was homologous since no change in stimulation by any other effectors was observed. Similar treatment of cyc- S49 cells, a mutant that lacks Gs, resulted in only a 10–15% receptor loss despite a 50% decrease in catecholamine stimulation of reconstituted adenylyl cyclase activity. By quantitative extraction of Gs from control and isoprenaline-treated WT cellmembranes and reconstitution into untreated cyc- cell membranes, it was demonstrated that the levels and behaviour of Gs in WT cell membranes were unchanged after isoprenaline treatment, despite a substantial loss of receptors from the cell surface. The β-adrenergic receptors lost from the cell surface could not be identified in any intracellular membrane fraction. Addition of cycloheximide to the culture medium of WT cells previously treated with isoprenaline resulted in a complete blockade of the restoration of β-adrenergic receptors which was otherwise observed within 16 h after removal of the agonist. It is concluded that, in the S49 cell line, prolonged exposure to agonists results in a loss of β-adrenergic receptors by a process dependent on the presence of Gs. However, the loss of receptors is selective since under conditions where the majority of receptors are lost, there is no measurable change in the level of cell surface membrane-bound Gs. The lost receptors appear to be degraded, since the restoration of receptor levels after removal of the agonist is dependent on protein synthesis.

In purified preparations of human erythrocyte GTP-binding proteins, we have identified a new substrate for pertussis toxin, which has an apparent molecular mass of 43 kDa by silver and Coomassie Blue staining. Pertussis toxin-catalyzed ADP-ribosylation of the 43-kDa protein is inhibited by Mg2+ ion and this inhibition is relieved by the co-addition of micromolar amounts of guanine nucleotides. GTP affects the ADP-ribosylation with a K value of 0.8 microM. Addition of a 10-fold molar excess of purified beta gamma subunits (Mr = 35,000 beta; and Mr = 7,000 gamma) of other GTP-binding proteins results in a significant decrease in the pertussis toxin-mediated ADP-ribosylation of the 43-kDa protein. Treatment of the GTP-binding proteins with guanosine 5'-O-(thiotriphosphate) and 50 mM MgCl2 resulted in shifting of the 43-kDa protein from 4 S to 2 S on sucrose density gradients. Immunoblotting analysis of the 43-kDa protein with the antiserum A-569, raised against a peptide whose sequence is found in the alpha subunits of all of the known GTP-binding, signal-transducing proteins (Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 265-259) showed that the 43-kDa protein is specifically recognized by the common peptide antiserum. A pertussis toxin substrate of similar molecular weight was observed in human erythrocyte membranes, bovine brain membranes, membranes made from the pituitary cell line GH4C1, in partially purified GTP-binding protein preparations of rat liver, and in human neutrophil membranes. Treatment of neutrophils with pertussis toxin prior to preparation of the membranes resulted in abolishment of the radiolabeling of this protein. From these data, we conclude that we have found a new pertussis toxin substrate that is a likely GTP-binding protein.

Treatment of MDCK cells with glucagon results in decreases in glucagon, NaF and prostaglandin E1-stimulated adenylyl cyclase activities, indicating the occurrence of a heterologous desensitization process. The extent of desensitization was time and glucagon concentration dependent. Maximal desensitization (30-50% decrease in stimulation by various effectors) was obtained by 4 h at 100 nM glucagon. Glucagon also induced homologous desensitization since after treatment, the Kact of glucagon was specifically increased. Treatment of cells with 10 microM 8-bromoadenosine 3':5'-monophosphate or 10 microM forskolin resulted in decreased hormonal (glucagon and prostaglandin E1) stimulation without any decrease in the stimulation by nonhormonal effectors (NaF, forskolin, and guanyl-5'-yl imidodiphosphate). The stimulatory regulator (Ns) of the adenylyl cyclase system was analyzed after desensitization with glucagon and no measurable changes in the apparent levels of the alpha s subunits of Ns or the activity of Ns as assessed by reconstitution of the cyc- S49 cell membrane adenylyl cyclase were detected. Levels of the alpha i subunit of the inhibitory regulator (Ni) were monitored by labeling with [32P]NAD and pertussis toxin. Membranes of glucagon-treated cells showed a 2-fold increase in the amount of alpha i labeled. Addition of pure Ns to glucagon-treated MDCK cell membranes restored full stimulation by NaF but did not restore stimulation by prostaglandin E1 or glucagon. It is concluded that glucagon induces heterologous and homologous desensitization of the MDCK cell adenylyl cyclase. The locus of the heterologous desensitization is at the level of the regulatory components. Decreased stimulation is thought to occur due to either an increase in the levels of Ni or due to altered interactions between the subunits of Ni.

The hepatic glucagon receptor was covalently labeled with [125I-Try10]monoiodoglucagon [( 125I]MIG) by use of the heterobifunctional cross-linker hydroxysuccinimidyl p-azidobenzoate. Labeling of the Mr = 63,000 peptide was sensitive to glucagon and GTP at concentrations at which they affect [125I]MIG binding to the receptor. The labeled receptor was solubilized with Lubrol-PX, and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are: S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f0 = 1.8, and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees C for 15 min prior to the addition of [125I]MIG permitted us to identify the high molecular weight form (Mr = approximately 113,000) of the receptor by direct sodium dodecyl sulfate-gel electrophoretic analysis. The Mr = 63,000 peptide can be adsorbed to wheat germ lectin-Sepharose. The glycoprotein nature of the receptor has been utilized to develop an assay for the detergent-solubilized receptor that uses wheat germ lectin-Sepharose as a solid matrix to adsorb the [125I] MIG-receptor complex. The free hormone remains in the liquid phase and is removed in the supernatant after low speed centrifugation. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) solubilizes receptors with retention of [125I]MIG binding activity. [125I]MIG binding to the CHAPS-solubilized receptor is specifically affected by unlabeled glucagon. Interaction of [125I]MIG with the soluble receptor is insensitive to the presence of GTP. IC50 for glucagon using the soluble receptor was 33-70 nM, irrespective of the presence or absence of GTP, while when the membrane-bound receptor was used, the IC50 in the absence of GTP was 2-4 nM and in the presence of GTP was 35-80 nM. These data allow us to conclude that the hepatic glucagon receptor in the membrane and in the nondenaturing detergent solution is a dimer of the Mr = 63,000 hormone-binding subunit and a glycoprotein. The soluble receptor does not display any functional interaction with the stimulatory regulator.

1. The effects of acute treatment of 3-methylcholanthrene (MC)-induced rats with carbon tetrachloride (CCl4) on the content and activity of the polycyclic aromatic hydrocarbon-inducible forms of cytochrome P-450 (P-450c and P-450d) in liver and kidney have been determined. 2. Post-treatment of MC-induced rats with CCl4 in vivo decreased the specific content of total, spectrally determined, P-450 in both hepatic and renal microsomes, by 60% and 40%, respectively. CCl4 treatment destroyed almost all of the hepatic P-450d (specific content after 6 h, less than 2% of control), but had no effect on P-450c, which increased slightly over the 6 h, to 30% above control values. 3. Immunocytochemical measurements demonstrated greater loss of P-450d from the centrilobular and midzonal than from periportal regions of the liver. 4. Hepatic phenacetin O-deethylase, an activity catalysed specifically by P-450d in this tissue, was dramatically decreased following administration of CCl4 to MC-induced rats. Loss of monooxygenase activity was highly correlated with the decrease in P-450d content (r = 0.947, P less 0.001). Aryl hydrocarbon hydroxylase activity of liver, catalysed almost entirely by P-450c, was unchanged and neither activity was affected in kidney. 5. Treatment of MC-induced rats with CCl4 causes a selective loss of hepatic P-450d and associated monooxygenase activities. Phenacetin O-deethylation is catalysed specifically by P-450d in liver, but not in kidney. The mechanism for this destruction of P-450d may be suicide activation of CCl4, but the rate of such activation appears to be much lower than with P-450b. Alternatively, P-450d may be particularly sensitive, and P-450c particularly resistant, to the active metabolite of CCl4 diffusing from a distant site of formation.

1. In the safety evaluation of drugs and other chemicals it is important to evaluate their possible inducing and inhibitory effects on the enzymes of drug metabolism. 2. While many similarities exist between species in their response to inducers and inhibitors, there are also important differences. Possible mechanisms of such variation are considered, with particular reference to the cytochrome P-450 system. 3. Differences in inhibition may be due to differences in inhibitory site of the enzyme involved, which is not always the active site of the enzyme, in competing pathways or in the pharmacokinetics of the inhibitor. 4. Differences in induction could be due to differences in the nature of the induction mechanism, in the isoenzyme induced, in tissue- or age-dependent regulation, in competing pathways for the substrate or its products, or in the pharmacokinetics of the inducing agent. 5. Examples of each of these possible differences are considered, often from our own work on the P450 IA subfamily, and results in animals are compared with those in humans, where possible. 6. At present, the differences between species in their response to inducers and inhibitors make extrapolation to humans from the results of animal studies difficult, so that ultimately such effects should be studied in the species of interest, humans.