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While the use of mandatory child restrainer car seats is evaluated by health professionals as in the domain of general public good, the implementation and prerequisite purchase may work a hardship on limited income individuals, especially those patients who deliver at city-county public hospitals. As legislation throughout the United States has begun to require mandatory car seat usage, the provision of such seats should also be addressed by public agencies who serve indigent populations. The purpose of this paper is to describe the establishment of an ongoing program for a lend-lease child restrainer car seat project in a public hospital setting. This paper also provides some suggestions to professionals who want to introduce and cultivate positive health care practices among populations which usually are non-responsive to primary prevention and health promotion.

Mammalian brain ribosomes were found to be heat-labile. On preincubation of the ribosomes at 37 degrees C, their ability to participate in polypeptide-synthesis reactions was substantially diminished. Despite the sensitivity of ribosomal protein synthesis to heat-inactivation, preincubation resulted in no significant alterations in ribosomal sedimentation profiles or changes in the integrity of the ribosomal RNA. The thermolability of brain ribosomes was shown to be associated with their inability to bind both template RNA and aminoacyl-tRNA. Similar experiments with brain ribosomal subunits demonstrated that the small (40S) subunit was more sensitive to heat-inactivation than the large (60S) subunit. The presence of ATP (1mm) protected ribosomes from thermal inactivation, although this protection was shown to be temporary. The protection appeared to be specific to nucleoside triphosphates, since GTP and UTP also stabilized ribosomes to thermal denaturation whereas nucleoside diphosphates (ADP) and nucleoside monophosphates (AMP and cyclic AMP) did not alter ribosomal sensitivity to heat. Although 1mm concentrations of nucleoside triphosphates protected ribosomes from heat-inactivation, the presence of higher concentrations resulted in complete inactivation of ribosomal activity.

The prevalence of Brucella canis agglutinins was determined in 170 cats (114 from animal shelters in California and 56 from an animal hospital in Texas). Seropositive reactions in the cats from animal shelters were 5.3, 11.4, and 0%, respectively, the the rapid slide agglutination test, salt 2-mercaptoethanol tube agglutination test, salt 2-mercaptoethanol tube agglutination test, and brucellosis card test. For hospitalized cats, the respective percentages were 7.1, 8.9, and 0%. One (0.9%) of 114 cats from the animal shelters and 5 (8.9%) of 56 hospitalized cats were seropositive by the salt 2-mercaptoethanol tube agglutination test at titers greater than or equal to 1:200. Isolation of bacteria was not attempted; thus, the findings of this study may need cautious interpretation.

The pathogenicity of infection produced following intracerebral (i.c.) inoculation of wild-type vesicular stomatitis virus (VSV) or temperature-sensitive (ts) mutants of VSV was compared. ts mutants used were ts 31 (VSV complementation group II) and ts 41 (VSV complementation group IV). The i.c. injection of wild-type VSV in weanling Swiss mice produced a rapidly fatal encephalitis with death of mice in 2 to 3 days. Histopathologically, such mice exhibited minimal changes of encephalitis on light microscopy. In contrast to the highly virulent, rapidly fatal central nervous system (CNS) infection seen after i.c. inoculation of wild-type VSV, infection with ts 31 VSV produced a more slowly progressive CNS infection characterized by hind limb paralysis and death 6 to 9 days after infection. Histopathologically, CNS infection with ts 31 is associated with previously unreported extensive spongiform changes in the gray matter of the spinal cord. The inoculation of ts 41 i.c., on the other hand, did not result in either clinical illness or histopathological changes in the spinal cords or brains of infected mice. The absence of clinical and histopathological lesions following i.c. infection of ts 41 VSV suggests that the capacity to alter the pathogenesis of VSV CNS infection may be a function of only certain ts mutants of VSV.

Mice infected intracerebrally (i.c.) with wild-type (wt) VSV or temperature-sensitive (ts) mutants, ts 11, ts 22, ts 31 and ts 41, were studied for the development of histopathological lesions in the central nervous system (CNS). Mice infected i.c. with wt VSV exhibited histopathological lesions consisting principally of occasional foci of perivascular mononuclear cell infiltration and rare foci of necrosis. All wt VSV infected mice died within 2 days of i.c. inoculation. In contrast, mice infected i.c. with ts 22 and ts 31 developed spongiform lesions limited to the grey matter of the spinal cord beginning 4 days after inoculation. The spongiform lesions rapidly spread to involve the entire grey matter of the spinal cord by 5-7 days after infection. Vacuolar changes were restricted principally to neuronal processes and astrocytes. Ts 22 and ts 31 infected mice developed neurological illness beginning 4 days after infection and the majority of mice died by 7 days after infection. Mice infected with ts 11 and ts 41, on the other hand, remained clinically well and were devoid of neuro-pathological lesions at 4 and 8 days after infection.

A sialoglycopeptide inhibitor, isolated from bovine cerebral cortex cells, that reversibly inhibits protein and DNA synthesis, was coupled to either Sepharose or polyacrylamide beads. Whereas over 1 ng of the inhibitor was released from Sepharose beads after 30 min at 37 degrees C, less than 0.2 ng of the sialoglycopeptide was released from the polyacrylamide beads. When added to 3T3 cells, the immobilized sialoglycopeptide efficiently inhibited protein synthesis. No detectable sialoglycopeptide inhibitor was released into the assay medium in the presence or absence of 3T3 cells. Addition of [125I]sialoglycopeptide, coupled to acrylamide P100 beads, to 3T3 cells also demonstrated that the sialoglycopeptide was not internalized by the cells. Thus we conclude that an interaction of the sialoglycopeptide at the cell surface is sufficient for biological inhibitory activity.

We have recently described the isolation and purification to homogeneity of a new sialoglycopeptide from bovine brain cell surfaces that reversibly inhibits protein synthesis and DNA synthesis of normal but not transformed cells. Active inhibitory preparations, however, were shown to contain a protease activity that was not lost upon purification. Several experiments were performed to establish the relationship between the proteolytic activity of the sialoglycopeptide and the biological inhibitory activity. Both the protease activity and inhibitory activity were stable at pH 6-8 but were reduced or completely destroyed below pH 4 and above pH 9. Acid inactivation was reversible and upon dialysis, both the biological inhibitory and protease activities were regained. Deglycosylation and CNBr cleavage indicated that the polypeptide backbone, rather than carbohydrate moiety, played an important role in the protease and biological inhibitory activities. Furthermore, chemical modification of amino and tyrosine groups indicated that both residues are essential for both activities. Thus, the biological inhibitory activity and protease activity are very closely related and most likely reside with the same polypeptide sequence.

A glycopeptide, isolated from bovine cerebral cortex cells and added in only nanogram levels to cells in culture, has been shown to inhibit both cell protein synthesis and cell division. When purified by gel filtration and Ulex europaeus lectin affinity chromatography, the radioiodinated preparation was subjected to high resolution isoelectric focusing and shown to contain three species of macromolecules. The glycopeptide focusing at pH 8.1 comprised over 75% of the radioiodinated material and possessed inhibitory activity against both cell protein synthesis and cell division. A second species that focused at pH 8.3 was also found to be inhibitory to cell metabolism and may have represented a variant of the major glycopeptide.

The ability of a naturally occurring cell surface sialoglycopeptide growth inhibitor to antagonize the induction of DNA synthesis by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied with mouse 3T3 cells. The bovine sialoglycopeptide was shown to be a potent antagonist of TPA-induced DNA synthesis in confluent 3T3 cell cultures. Kinetic studies demonstrated that inhibition of TPA-induced DNA synthesis required the addition of the sialoglycopeptide within 15 min of TPA treatment. Addition of the sialoglycopeptide 30 min or longer after the cells were exposed to TPA did not block stimulation of DNA synthesis by TPA. The inhibition of TPA action was shown not to be restricted to DNA synthesis in 3T3 cultured cells since the sialoglycopeptide also inhibited TPA-induced ornithine decarboxylase (ODC, L-ornithine carboxylase, EC 4.1.1.17) activation in suspensions of mouse epidermal and 3T3 cells.

This study assessed the knowledge and usage patterns of vaginal douching in sexually active teenagers attending a family planning clinic. A questionnaire was administered consecutively to 94 black, 36 Hispanic, and 12 Anglo females ranging in age from 13 to 19 years. The survey showed that vaginal douching is a common practice, with almost two thirds learning about douching from their mother. The technique is primarily used for hygienic reasons with few using it to prevent pregnancy or infection. Age of first douche correlated with age of first intercourse (p less than 0.001). Almost 23% had douched within 2 days of the clinic visit, and 56% reported douching one or more times a week.

Morphological changes were extensive following infection of murine neuroblastoma N-18 cells with a temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV), G31 (complementation group III), and incubation at 39 degrees C, a non-permissive condition for virion maturation. Incubation for 24 h after infection resulted in extensive morphological degeneration of mitochondria with over 80% from that in uninfected cells. Janus green B supravital staining, was reduced by 81% from that in uninfected cells. Cellular ATP levels were reduced by 50% 12 h after infection. Mitochondrial degeneration still occurred in infected cells after the inactivation of lysosomes with chloroquine. Extensive cell fusion and cytoplasmic vacuole formation also occurred during the non-permissive infection with ts G31. Loss of plasma membrane integrity was not the cause of vacuole formation since 90% of the cells were able to exclude trypan blue 24 h after infection, nor were the vacuoles the result of inactivation of the mitochondria since cyanide-poisoned cells did not form vacuoles. The cytopathic alterations observed in N-18 cells during the non-permissive infection of N-18 cells with ts G31 did not occur during the non-permissive infection of N-18 cells with ts G11 (I), ts G41 (IV), or u.v.-inactivated ts G31. However, the non-permissive infection with ts O45 (V) led to mitochondrial degeneration and cytoplasmic vacuole formation, but no cell fusion occurred. These results are discussed in light of the ultrastructural features previously observed in the central nervous system of mice infected with ts G31 and cells in culture infected with wild-type VSV.

Outbred Swiss mice 3 to 4 weeks of age were injected intracerebrally with wild-type vesicular stomatitis virus or its temperature-sensitive (ts) mutants ts 11, ts 22, ts 31, and ts 41. Brain and spinal cord were then studied for pathologic changes by electron microscopy. All mice infected with wild-type vesicular stomatitis virus died within 2 days of inoculation. Diffuse ependymal alterations often culminating in necrosis in brain and especially spinal cord and rare foci of necrosis and mononuclear cell infiltration in the injected hemisphere were the main pathologic changes in these animals. In contrast, mice infected intracerebrally with ts 22 and ts 31 showed their first clinical signs, consisting of hind limb paralysis, on day 4 and did not die until day 7 or 8 after inoculation. Ependymal alterations were of less severe degree in these animals, whereas the most striking changes were those of status spongiosus limited to the gray matter of the spinal cord. Such status spongiosus was mainly due to ballooning of dendrites and astrocytic processes, although myelin and neurons were also occasionally involved. Mice infected with ts 11 and ts 41, on the other hand, remained clinically well and failed to show significant pathologic features at 4 and 8 days after intracerebral inoculation. This study would indicate that some ts mutants of vesicular stomatitis virus are capable of altering the fulminating disease produced by the parent virus and of producing strikingly different pathologic changes.

Inoculation of wild-type (wt) VSV intracerebrally (i.c.) in Swiss weanling mice results in a rapidly fatal illness with death in two to three days. In contrast, i.c. inoculation of temperature-sensitive (ts) VSV mutants G3I and G22, but not ts GII or ts G4I, results in a more slowly progressive central nervous system (CNS) disease with distinct neurological signs. Studies undertaken to evaluate the neurovirulence of ts VSV mutants indicated that the ability of ts mutants to produce pathological changes in the CNS of mice appeared related to their ability to replicate to high titre in brain and spinal cord. However, replication of ts VSV mutants in brain alone was not sufficient to produce clinical illness. More importantly, the ability of ts VSV mutants to replicate at non-permissive temperatures in vitro did not appear to correlate with neurovirulence. VSV harvests from brains and spinal cords of mice infected with each of the ts mutants were temperature-insensitive. In spite of their temperature-insensitivity, the biological behaviour of viruses recovered from CNS tissues was, surprisingly, not that which was characteristic of revertant clones. Virus isolates recovered from infected CNS tissues, despite their temperature-insensitivity, behaved biologically like the orignal stocks of ts mutant virus. These data suggest that temperature-sensitivity is not directly correlated with the unique pathogenesis elicited by infection with ts VSV mutants.

A novel glycopeptide inhibitor of cell division, isolated from bovine cerebral cortex cell surfaces, was shown to selectively protect nonmalignant cells from the cytoxic action of 5-bromo-2-deoxyuridine (5-BrdUrd). When mouse LM-22 cells (nonmalignant and devoid of gangliosides) were preincubated with GM1 ganglioside (3.0 micrograms/ml), the cell surface glycopeptide inhibitor effectively arrested cell division. In contrast to LM-22 cells, transformed mouse fibrosarcoma (No. 1316) cells were insensitive to the glycopeptide inhibitor whether or not they were preincubated with GM1 ganglioside. Mixed cultures of LM-22 cells preincubated with GM1 ganglioside and 1316 fibrosarcoma cells at an approximate ratio of 1:1 were established. Since LM-22 cells are resistant and 1316 fibrosarcoma cells are sensitive to 3.0 mM ouabain, the identity of surviving cells following BrdUrd treatment could easily be determined. Three hr after the establishment of the mixed cell population, 250 ng protein per ml of the purified bovine glycopeptide inhibitor was added to selectively arrest the mitosis of the LM-22 cells. After an additional 3 hr of incubation, 5-BrdUrd was added to a final concentration of 5.0 mM. Twelve hr later, cells were serially diluted and seeded into duplicate plates with and without 3.0 mM ouabain. LM-22 cells were effectively protected from the cytotoxic action of 5-BrdUrd (92 to 94% survival) while the majority of the 1316 fibrosarcoma cells were killed (21 to 30% survival). The selective protection of LM-22 cells was shown to be independent of differences in plating efficiency, cytotoxicity of 5-BrdUrd in the absence of the glycopeptide inhibitor, and the generation time of the two cell lines.

In previous reports, we have identified cell-surface glycopeptides from mouse cerebrum (BCSG) that inhibited protein synthesis and mitosis in several cell types. When baby hamster kidney (BHK)-21 cells were infected with vesicular stomatitis virus (a negative strand RNA virus), BCSG extensively inhibited viral protein synthesis. This inhibition was effective against both protein and glycoprotein synthesis and was independent of amino acid uptake by infected cells, synthesis of viral RNA, and degradation of viral proteins. Analysis of polyribosome profiles in uninfected BHK-21 cells indicated that the degree of cellular protein synthesis inhibition could not be attributed to activation of RNase or solely to a disruption of chain initiation. When added directly to a cell-free protein-synthesizing system derived from BHK-21 cells, BCSG was ineffective, but if the inhibitory material was first allowed to react with cells, cell-free protein synthesis was substantially reduced. When BCSG were reacted with cells for 5 min at 0 degrees C, the cells tested, BHK-21 (a BCSG-sensitive line) and murine fibrosarcoma 2237 (a BCSG-insensitive line), both effectively adsorbed the inhibitor from the medium.

We have isolated from bovine cerebral cortex cells and purified to homogeneity an 18,000 dalton, pl 3.0 sialoglycopeptide that inhibits protein synthesis and DNA synthesis of nontransformed but not transformed cells without affecting uptake of radiolabeled precursors. In this paper, we examine the relationship between the binding of the sialoglycopeptide inhibitor to 3T3 cells and inhibition of protein synthesis. Binding of the sialoglycopeptide to 3T3 cells was rapid at 37 degrees C and reached a maximum at 30 min; the binding at 37 degrees C was shown to be saturable and specific. Scatchard analysis of the binding indicated that 3T3 cells contained about 2 X 10(4) receptors/cell with a dissociation constant of 1.0-1.5 nM. Several lines of evidence indicated that receptor occupancy on 3T3 cells correlated with the protein synthesis inhibitory activity of the sialoglycopeptide. A comparison of the kinetics of inhibitor binding with the kinetics of protein synthesis inhibition demonstrated that binding directly correlated with the inhibition of protein synthesis, concentration-dependent inhibition of protein synthesis directly correlated with concentration-dependent receptor occupancy, and a direct correlation was also observed between the kinetics of inhibitor dissociation from its specific cell surface receptor and the kinetics of recovery from protein synthesis inhibition.

We have isolated and characterized glycopeptides, derived from mouse and bovine cerebral cortex cells, that inhibit protein synthesis and cell growth of normal but not transformed cells. The inhibitor binds to target cell surfaces, and gangliosides have previously been shown to influence cell sensitivity to the glycopeptides. Preincubation with 3.0 micrograms/ml ganglioside GM1 at 0 degrees C for 3 hr sensitized the mouse L-cell line to the inhibitor, as determined by protein synthesis assays. Preincubation of LM cells with ganglioside GM1 alone did not affect protein synthesis rates. In addition, the gangliosides GD1a and GM3 also sensitized the LM cells to the protein synthesis inhibitory effect of the glycopeptide inhibitor. Binding experiments were performed with 3T3 (sensitive) and LM (insensitive) cells to determine if sensitivity to the glycopeptide inhibitor was reflected in binding of the inhibitor to these cells. Binding of 125I-labeled inhibitor to 3T3 cells was maximal after 60 min at 0 degrees C and saturable at approximately 1 X 10(4) molecules/cell. Furthermore, binding of the inhibitor was dose-dependent, with half-maximal binding at 1.5-2.0 nM and saturation at 8.0-10.0 nM. Scatchard plot analysis indicated that the Kd was about 1 X 10(-9) M and that there are 1 X 10(4) receptors/cell. Binding of the inhibitor to LM cells was maximal after 30 min at 0 degrees C and saturation occurred at 5 X 10(3) molecules/cell. We then examined the possibility that gangliosides are the cellular receptor or co-receptor for the glycopeptide inhibitor. Binding of the inhibitor to ganglioside GM1 was first examined after the ganglioside had been preadsorbed to polystyrene tubes. These experiments indicated that the ganglioside did not bind the inhibitor. Ganglioside-containing liposomes from phosphatidylcholine or LM cell membrane components were also prepared; these artificial membranes did not bind appreciable amounts of the iodinated inhibitor. Competition experiments showed that the gangliosides GM1 and GD1a did not neutralize the protein synthesis inhibitory activity of the glycopeptides, indicating that gangliosides do not directly interact with the glycopeptide inhibitor. In addition, binding of the inhibitor to LM cells preincubated with ganglioside GM1 was studied. Although the binding of the inhibitor to LM cells was one-half that observed for 3T3 cells, incorporation of exogenous gangliosides into LM cells did not result in increased binding of the inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)

A temperature sensitive mutant of vesicular stomatitis virus which does not mature properly when grown at 39 degrees C promoted extensive fusion of murine neuroblastoma cells at this nonpermissive temperature. Polykaryocytes apparently formed as a result of fusion from within the cells that requires low doses of infectious virions for its promotion and is dependent on viral protein synthesis. Although 90% of infected N-18 neuroblastoma cells were fused by 15 h after infection, larger polykaryocytes continued to form, leading to an average of 28 nuclei per polykaryocyte as a result of polykaryocytes fusing to each other. Two neuroblastoma cell lines have been observed to undergo fusion, whereas three other cell lines (BHK-21, CHO, and 3T3) were incapable of forming polykaryocytes, suggesting that nervous system-derived cells are particularly susceptible to vesicular stomatitis virus-induced fusion. Although the normal assembly of the protein components of this virus is deficient at 39 degrees C, the G glycoprotein was inserted into the infected cell membranes at this temperature. Two lines of evidence suggest that the expression of G at the cell surface promotes this polykaryocyte formation: (i) inhibition of glycosylation, which may be involved in the migration of the G protein to the cellular plasma membranes, will inhibit the cell fusion reaction; (ii) addition of antiserum, directed toward the purified G glycoprotein, will also inhibit cell fusion.

While the seriousness of sexual abuse by adolescents is finally beginning to receive adequate attention from the professional community, the existence of child perpetrators is largely dismissed and denied. Forty-seven boys between the ages of 4 and 13 are described who have molested children younger than themselves. Coercion was involved in all of the cases included in this study. These children had been treated in a program especially designed for child perpetrators at Children's Institute International in Los Angeles. Prior to their own sexually abusive behaviors, 49% of these boys had been sexually abused and 19% physically abused. The children all knew the people who victimized them. These male child perpetrators all knew the children they molested. In 47% of the cases the sexual abuse was of a sibling. The average number of victims of these children was 2.1 with a range of 1 to 7. The mean age at the time of perpetration was 8 years, 9 months. The mean age of the victims of these children was 6 years, 9 months. There was a history of sexual and physical abuse in the majority of the families of these children, as well as a history of substance abuse. This population is compared to adolescent perpetrators.

Gradient SDS-polyacrylamide gel electrophoresis (PAGE) and proteolytic digestions were utilized to examine the virion proteins of two isolates of wild-type vesicular stomatitis virus (WT-VSV), WTATCC from the American Type Culture Collection and WTGL and Glasgow, as well as temperature-sensitive (ts) mutant ts G31 and a central nervous system (CNS) isolate of ts G31 designated ts G31BP. The WTATCC M protein differed in electrophoretic mobility and in its tryptic or chymotryptic peptide maps from the 125I-labelled M proteins in WTGL, ts G31 or ts G31BP. The M protein in the latter three viruses appeared identical using either tryptic or chymotryptic digestion procedures; however, limited digestion with V8 protease revealed a difference between the M protein of ts G31 and both WTGL and ts G31BP M proteins. The L, NS and G proteins all had identical tryptic and chymotryptic peptide maps in WTGL, ts G31 and ts G31BP virions. The N protein, however, was demonstrated to be distinctly different in the WTGL virion when compared with the ts G31 (or ts G31BP) virion by its tryptic peptide map. In addition, limited proteolytic digestion of the 125I-labelled N proteins revealed a different peptide structure in ts G31BP compared to N proteins of ts G31 or WTGL. The altered N protein in the CNS isolate, ts G31BP, is discussed in terms of its altered in vivo phenotype of labile viral RNA, and its potential role in the unique CNS disease associated with this virus.

Glycopeptides were isolated from the cell surfaces of mouse brain cortical tissue that inhibited both cell division and protein synthesis by cells in culture. The protein-synthesis inhibition appeared to affect most cells exposed and was equally effective against glycoprotein and protein synthesis. The inhibition of protein metabolism was independent of mRNA synthesis and uptake of labelled precursors into intracellular pools, indicating that it was directed at intracellular translational events. Fractionation of chloroform/methanol-extracted preparations of this brain cell-surface substance on Bio-Gel P-100 revealed the material to be quite heterogenous, although inhibitory activity was found only in fractions of mol.wt. 25000--30000 and 6000--10000. Biochemical analysis of these fractions demonstrated that they were 6% carbohydrate and 94% amino acid by weight. The 25000--30000-mol.wt. glycopeptides were shown to inhibit cell growth at concentrations of 2 microgram/ml in cultured cells and to inhibit protein synthesis by 50% at concentrations of 3 microgram/ml. The 25000--30000-mol.wt. brain-cell-surface-substance glycopeptides were further purified by ultrafiltration and affinity chromatography with Ulex europaeus agglutinin, resulting in a 400-fold increase in specific biological activity. The inhibitor was not lethal to cells and was not species- or tissue-specific.

The effect of a single injection of phenylalanine (2 mg/g of body weight) on brain polyribosomes, which increases the number of inactive monoribosomes, persists for 2 to 3 hours. A single injection of seven large neutral amino acids after phenylalanine administration results in a reversal of the effect on brain polyribosomes with a resultant decrease in monoribosomes to near normal levels. The other common amino acids are apparently not limiting during hyperphenylalanemia, because an injection of these did not increase recovery.

An acute administration of phenylalanine to neonatal animals has been reported to result in large decreases in the intracellular concentrations of several essential amino acids in neural tissue, as well as an inhibition of neural protein synthesis. The present report evaluates the effects of the loss of amino acids on the concentrations of aminoacyl-tRNA in vivo, with the view that an alteration in the concentrations of specific aminoacyl-tRNA molecules could be the rate-limiting step in brain protein metabolism during hyperphenylalaninaemia. tRNA was isolated from saline- and phenylalanine-injected mice 30-45 min after injection, by using a procedure designed to maintain the concentrations of aminoacyl-tRNA present in vivo. Periodate oxidation of the non-acylated tRNA and aminoacylation with radioactively labelled amino acids was used to determine the proportion of tRNA that was present in vivo as aminoacyl-tRNA. Although decreases in the intracellular concentrations of alanine, lysine and leucine were observed after phenylalanine administration, the concentrations of alanyl-tRNA, lysyl-tRNA and leucyl-tRNA actually increased by 15%. Although tryptophan has been suggested to be rate-limiting during hyperphenylalaninaemia, the proportion of tryptophan tRNA that was acylated was maximal in both normal and hyperphenylalaninaemic animals. This unexpected increase in aminoacyl-tRNA concentration is discussed as perhaps a secondary effect resulting from the phenylalanine-induced inhibition of protein synthesis. In contrast, the proportion of methionine tRNA that was acylated in vivo after phenylalanine administration was demonstrated to be decreased by approx. 17%. When the isoaccepting species of methionine tRNA were separated by reverse-phase column chromatography, three species were separated, one of which was demonstrated to be the initiator species, tRNAfMet, by the selective aminoacylation and formylation with Escherichia coli enzymes. After the administration of phenylalanine, the acylation of each of the three methionine tRNA species was decreased, with the initiator species being lowered by 10%. This effect on aminoacylation of tRNAfMet may be the primary step by which phenylalanine affects neural protein synthesis, and this is consistent with previous reports that re-initiation may be inhibited during hyperphenylalaninaemia.

An 18-kDa 125I-sialoglycopeptide growth inhibitor was covalently cross-linked to its binding site on intact cultured Swiss 3T3 cells by three bifunctional cross-linkers with short (dimethyl adipimate), medium (disuccinimidyl suberate), and long (bis(2-succinimidooxycarbonyloxyethyl)sulfone) chain lengths. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated a band of Mr approximately 168,000 regardless of which cross-linker was used. The labeling of this band was specific in that it was prevented by excess unlabeled inhibitor and the apparent molecular weight of the cross-linked receptor-ligand complex was unchanged by treatment with reducing agent. The efficiency of the cross-linking was increased by increasing pH, and the extent of covalent cross-linking was dependent on the concentration of the bifunctional reagent. Octyl glucoside and sodium dodecyl sulfate were effective in solubilizing the receptor while Triton X-100 did not extract the receptor from the plasma membrane. These observations suggest that the 168-kDa binding species represents the 125I-sialoglycopeptide cross-linked to a specific plasma membrane receptor and that the receptor does not appear to contain interchain disulfide bonds.

We have isolated and purified a cell surface sialoglycopeptide (SGP) from bovine cerebral cortex cells that previously was shown to be a potent inhibitor of cellular protein synthesis. The following studies were carried out to characterize the potential ability of the SGP to inhibit DNA synthesis and to arrest cell division. Treatment of exponentially proliferating Swiss 3T3 cells with the SGP inhibitor resulted in a marked inhibition of thymidine incorporation within 24 h. When the SGP was removed from inhibited cultures, a sharp rise in 3H-thymidine incorporation followed within 3-4 h that peaked well above that measured in exponentially growing cultures, suggesting that the inhibitory action of the SGP was reversible and that a significant proportion of the arrested cells was synchronized in the mitotic cycle. In addition to DNA synthesis, the inhibitory action of the SGP was monitored by direct measurement of cell number. Consistent with the thymidine incorporation data, the SGP completely inhibited 3T3 cell division 20 h after its addition to exponentially growing cultures. Upon reversal there was a delay of 15 h before cell division resumed, when the arrested cells quickly doubled. Most, if not all, of the growth-arrested cells appeared to have been synchronized by the SGP. The SGP inhibited DNA synthesis in a surprisingly wide variety of target cells, and the relative degree of their sensitivity to the inhibitor was remarkably similar. Cells sensitive to the SGP ranged from vertebrate to invertebrate cells, fibroblast and epitheliallike cells, primary cells and established cell cultures, as well as a wide range of transformed cell lines.

A kinetic analysis of the appearance of 14C-labelled proteins in the surface membranes isolated from exponentially growing neuroblastoma cells (N2a) showed that the total membrane proteins reached a steady-state specific radioactivity in 18-20 h. However, examination of individual protein bands resolved by sodium dodecyl sulphate-urea-polyacrylamide-gel electrophoresis illustrated that differences in the kinetics of specific surface-membrane proteins could be detected. Although most of the protein bands reached a steady-state specific radioactivity at a time similar to that for total membrane proteins, at least two bands (mol. wt. 180000 and 130000) attained the steady-state within 8-10 h. It was shown by the use of dual-labelling techniques that these two protein bands turned over in the surface membranes of neuroblastoma N2a cells at least 180 and 150% faster than the total membrane protein. These two proteins were glycosylated and located on the outer surface of the cells, since they were labelled with radioactive carbohydrates and readily removed by treatment of the intact neuroblastoma cell with proteinases.

A single injection of the hypothermia-inducing neuropeptide bombesin resulted in an excellent recovery system for reisolating viruses from Swiss albino mice infected with vesicular stomatitis virus even up to 90 days after infection. The virus was recovered from a cell homogenate prepared from whole brain tissue 24 h after intracerebral injection of bombesin; brain cells were cocultivated with BHK-21 cell monolayers and then plaqued on BHK-21 cells at 31 degrees C. All of the recovered viruses were identified as vesicular stomatitis virus by antibody neutralization and peptide analyses of some of the structural proteins. However, some of the recovered viruses were altered with regard to tryptic peptide maps, temperature sensitivity, and central nervous system disease induced compared with the viruses used to initiate the infection. Most of the recovered viruses induced a similar disease when reinoculated intracerebrally into mice, characterized by hind-leg paralysis 4 to 6 days after infection. Two of the recovered viruses were lethal, however, resulting in a relatively rapid generalized wasting disease and death in 3 to 4 days.

In contrast to wild-type vesicular stomatitis virus (VSV), which produces a fulminant illness with death in two to three days, mixtures of homologous defective autointerfering (DI) particles and wild-type VSV (DI-wild-type VSV) injected intracerebrally into mice resulted in a slowly progressive disease of the central nervous system (CNS). In fact, mice inoculated with DI-wild-type VSV survived up to nine days after infection and displayed striking paralysis of the hind limbs. The slowly progressive CNS disease accompanying infection with DI-wild-type VSV was characterized by production of only 1% of the amount of VSV recovered when wild-type VSV was injected alone. Morphologically, mice infected with DI-wild-type VSV displayed a spectrum of pathologic changes not encountered with disease due to wild-type VSV alone. These changes included the presence of parenchymal necrosis in the spinal cord, changes in and secondary demyelination of the white matter, striking leptomeningeal inflammation, and spongiform changes in the gray matter of the neuropil. Since these pathological features are not found when CNS disease results from either wild-type VSV or temperature-sensitive (ts) mutants of VSV, it is suggested that CNS infection with ts VSV is not mediated principally by production of DI particles. Furthermore, in vivo CNS infection with DI-wild-type VSV did not appear to be mediated by production of ts VSV mutants.

Mouse brain ribosomes were radioactively labelled by a cell-free reductive alkylation reaction with NaBH4 and [14C]formaldehyde. The radioactivity was largely associated with ribosomal proteins, but little, if any, of the rRNA was radioactive after the alkylation procedure. Both ribosomal structural proteins and loosely associated components were successfully labelled by this procedure. The sedimentation properties of the ribosomes were unaltered and their ability to carry out poly(U)-directed protein synthesis, although decreased, was largely retained. Incubation of 14C-labelled ribosomes with brain cytosol resulted in a 17% loss of radioactivity, although treatment of the ribosomes with 1.0 m-KCl to remove the loosely associated factors rendered the ribonucleoprotein particles resistant to cytosol effects. The ribosome-cytosol interactions did not appear to be related to an exchange process, since the released radioactivity was largely degraded to acid-soluble material. In addition, the incubation of native ribosomes with brain cytosol resulted in an almost complete loss in the ability of the ribosomes to participate in cell-free protein synthesis.

The control of cell proliferation involves the complex interaction between growth factors and growth inhibitors. We have examined this interaction with the mitogen epidermal growth factor (EGF) and a recently purified 18 kD, pI 3, sialoglycopeptide that reversibly inhibits cellular metabolism of a variety of cells. The sialoglycopeptide was a very potent inhibitor of EGF action; 0.22 nM of the inhibitor completely blocked the mitogenic effect of 1.60 nM of EGF. The sialoglycopeptide, however, did not affect the binding of EGF to 3T3 cells. Neither the mixed affinities (0.11-1.9 nM) of binding nor the total number of receptors (50,000 receptors/cell) for EGF were altered by the addition of the sialoglycopeptide. In addition, competitive binding experiments demonstrated the specificity of inhibitor binding to 3T3 cells and also showed that EGF and the sialoglycopeptide did not share the same receptor, suggesting that the inhibitor blocked EGF action at a postreceptor, intracellular event in the signal cascade. We further demonstrated that the sialoglycopeptide had to be added within 2.5 hr after EGF to block effectively the stimulation of DNA synthesis by the growth factor, suggesting that the inhibitor blocked EGF stimulation at a relatively early step in the signal transduction mechanism.

In order to resolve the functional role of intact rRNA in polypeptide chain elongation mouse brain ribosomes were treated with dilute pancreatic or T(1) RNAase (ribonuclease). After RNAase treatment, several physical-chemical properties as well as the functional activity of the ribosomes were measured. RNAase treatment resulted in the extensive hydrolysis of both 18S and 28S rRNA; however, the sedimentation properties of mono-ribosomes were unaltered and more than 90% of the relatively low-molecular-weight RNA fragments remained associated with ribosome particles. Analysis of the ability of RNAase-treated ribosomes to participate in cell-free protein synthesis showed that ribosomes with less than 2% intact rRNA retained more than 85% of their activity in polyphenylalanine incorporation. Proof that the incorporation of phenylalanine by ribosomes with hydrolysed rRNA actually represented active translocation was obtained by the effective inhibition of incorporation by diphtheria toxin. In addition, the oligopeptide products of protein synthesis could be identified by BD (benzoylated diethylaminoethyl)-cellulose column chromatography. Analysis of the size distribution of oligopeptides synthesized by normal and RNAase-treated ribosomes showed no significant differences which indicated that there was no change in the proportion of ribosomes engaged in protein synthesis. Thus strong RNA-protein and protein-protein interactions must serve to maintain the functional integrity of ribosomes even when the rRNA is extensively degraded. The ability of the enzyme-treated ribosomes to efficiently incorporate amino acids clearly demonstrated that ;intact' rRNA is not required for protein-synthetic activity.

A chronic hyperphenylalanemia was effectively produced in developing mice by daily administrations of phenylalanine (2 mg/g body wt) and a phenylalanine hydroxylase inhibitor alpha-methyl-D,L-phenylalanine (0.43 mg/g body wt). The presence of alpha-methylphenylalanine in newborn mice inhibited 65-70% of hepatic phenylalanine hydroxylase activity within 12 h. Since this maximum inhibition persisted for 24 h or longer, decreased enzyme activity was maintained by daily administrations. Whereas concentrations of phenylalanine increased approximately 40-fold in both plasma and brain following injection of alpha-methylphenylalanine and phenylalanine, plasma levels of tyrosine were not altered significantly. Concomitant with changes in phenylalanine concentrations we observed the brain polyribosomes' disaggregation, which reached a maximum 3 h after injection and persisted as long as 18 h. Polyribosomes did not become refractory to as many as 10 daily injections of alpha-methylphenylalanine and phenylalanine. In addition to polyribosome disaggregation, chronic hyperphenylalanemia reduced the rates of polypeptide chain elongation on polyribosomes isolated from brain homogenates.

A single intracerebroventricular injection of 100 ng of beta-endorphin altered the course of the central nervous system (CNS) infection of a temperature-sensitive mutant of vesicular stomatitis virus (VSV), tsG31-KS5. When mice were administered beta-endorphin and then 24 h later infected intracerebrally with tsG31-KS5 VSV, 70% of the animals died within 8 days of infection. In comparison, less than 10% of the animals had died after 21 days when infected with tsG31-KS5 VSV alone. When mice were injected with beta-endorphin and tsG31-KS5 VSV simultaneously, or with beta-endorphin 21 days after infection, the more aggressive clinical disease was not observed. Superficially, the more lethal disease induced by beta-endorphin appeared to be a result of a mild hypothermia caused by the neuropeptide. beta-Endorphin, however, did not influence the disease in nude (nu/nu) mice even though their core temperatures were reduced to an extent similar to that of BALB/c (+/+) mice, implicating the involvement of T lymphocytes in the alteration of the course of infection in normal mice.

The ability of the calcium ionophore A23187 and the sodium ionophore Monensin to antagonize the inhibition of 3T3 cell protein synthesis by a bovine cell surface sialoglycopeptide was measured. A23187, when added before and shortly after the sialoglycopeptide, significantly reduced the biological activity of the inhibitory glycopeptide. In contrast, Monensin had little, if any, influence on protein synthesis inhibition by the sialoglycopeptide. The ability of A23187 to circumvent the inhibitory action of the bovine glycopeptide was shown to be independent of the time the ionophore was incubated with the cells and the binding of the sialoglycopeptide to the 3T3 target cells. Neither the total amount of sialoglycopeptide bound to the cells, nor its affinity to the cell surface receptor, was influenced by the presence of A23187.

A bovine sialoglycopeptide, purified to homogeneity and capable of inhibiting cellular protein synthesis and proliferation, was shown to agglutinate a wide variety of nontransformed and transformed cells. The cell agglutination activity was shown to be independent of the biological inhibitory action and most likely related to a protease activity that could not be physically separated during purification of the sialoglycopeptide. Samples that were completely biologically inactivated retained full protease activity and their ability to agglutinate target cells. Balb/c 3T3 cells were not agglutinated by the sialoglycopeptide and they elicited a protein that interfered with the agglutination reaction and even redispursed cells that already had been aggregated by the inhibitor.

Inoculation of three- to four-week-old BALB/c mice with temperature-sensitive (ts) vesicular stomatitis virus (VSV) mutant G41 produced a subacute neurological disease mainly localized in the spinal cord. Meningitis and diffuse microglial infiltration of the anterior horns of the spinal cord were seen starting six days after infection when neuronal degenerative changes could be seen. Infection of neurons was demonstrated by immunofluorescence microscopy five days after infection. Two to three weeks after infection, loosening of the neuropil was evident due to neuronal dropout, and the mononuclear infiltration had become perivascularly distributed and had changed in character because of a striking increase in plasma cells. These cells together with Russell bodies became the main inflammatory cellular component about four to five weeks after viral inoculation. Starting eight days after infection, several foci of primary demyelination could be found in the anterior columns of the spinal cord. Immunological responses appeared within four days after infection when both neutralizing antibody and stimulation of specific spleen lymphocytes could be demonstrated. Serum antibody responses peaked at 21-28 days but remained elevated for up to 153 days. Stimulation of spleen lymphocyte cells peaked at 10-21 days and also remained elevated for as long as 116 days. The presence of both inflammatory changes and immunological responses to VSV mutant ts-G41 for prolonged periods is characteristic of persistent viral infections. Infection of BALB/c mice with ts-G41 thus represents the first in vivo example of persistent viral infection utilizing ts mutants of VSV.

Six temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV) isolated from the central nervous system (CNS) following injection with ts G31 (III) all possessed a post-transcriptional defect, not found in the initial virus, that affects the stability of viral RNA transcripts. Examination of viral RNA metabolism in mouse neuroblastoma (N-18) cells revealed that RNA synthesis of the CNS isolates was decreased considerably at elevated temperatures (up to 80 or 90% at 39 degrees C). In addition, analysis of the RNA transcripts suggested that little if any normal-sized transcripts were made in cells infected with these CNS isolates at either 37 degrees C or 39 degrees C. The RNA deficiencies did not appear to be the result of a temperature-sensitive lability of virion transcriptase as examined by in vitro transcriptase assays. However, when N-18 cells infected with one of the CNS isolates, ts G31 BP, were first preincubated at the permissive temperature of 31 degrees C for 3 h and then shifted to 39 degrees C, RNA synthesis proceeded at a rate comparable to that of 31 degrees C. The viral mRNA species synthesized following the temperature shift also contained normal sized tracts of poly(A) RNA, suggesting that neither the viral transcriptase nor its polyadenylate synthetase was thermally labile. However, for any of the six CNS isolates, all species of viral RNA synthesized in cells that were first preincubated at 31 degrees C degraded rapidly when the cells were shifted to 39 degrees C. In contrast little or no RNA degradation of either 42S progeny RNA or mRNA species was detected in the wild-type VSV, ts G31 or three other VSV mutants that are defective in some aspect of viral RNA metabolism: [ts G11 (I), ts G22 (II), ts G41 (IV)]. The apparent phenotype alteration in the stability of viral RNA in all of these CNS isolates is discussed in terms of the possible genotypic changes that may have occurred as well a the unique CNS disease that accompanies infection by these viruses.

Previous studies on vesicular stomatitis virus (VSV) maturation in infected cells have utilized in vitro cell cultures. The present study is, to our knowledge, the first in vivo analysis of VSV-cell interaction in the central nervous system of weaning outbred Swiss mice. Intracerebral inoculation of wild-type VSV resulted in rapid viral replication in brain and spinal cord. By immunoflourescence, viral antigens were first seen in ependymal cells of brain and spinal cord and soon thereafter in surrounding neurons. The large anterior horn neurons of spinal cord appeared to be in the most heavily infected. Ultrastructurally, VSV-neuron interaction evolved in three phases. The first phase consisted of viral entry into the cell by fusion and viropexis. The second phase was characterized by nucleocapsid accumulation and resulted in the appearance of large cytoplasmic inclusions. The third phase was maturation and release from the infected cell and was accomplished by viral budding from plasma membranes. Degenerative changes in infected neurons were generally absent. Cells in the area of the central canal seemed to present a different pattern of virus-cell interaction especially at the level of maturation and release. Some of these cells in advanced stages of degeneration showed viral particles free in nucleocapsid material with no virus-membrane association. Viral budding was not observed and, because these cells do eventually die, it is possible that virus was released in the intercellular space at the moment of cellular disruption. These results suggest that VSV-cell interactions may vary depending upon the nature of the infected cells.

One hundred and four indigent, pregnant adolescents were selected to evaluate the effect of a prenatal education curriculum presented in a maternity clinic. Adolescent mothers were visited 6 months postpartum to test which short-term, intermediate, and long-term curriculum content items were accurately and effectively retained. Success of the curriculum was evaluated by a trained social worker in the girls' home to ascertain how these content areas were reflected in the adolescent mother's behavior. Short-term matter-child health-care compliance content showed a high degree of success. Intermediate child-rearing content techniques often did not lead to appropriate behavior six months after the baby was born. Long-term educational and vocational performance content did not demonstrate effective long-term retention. Technical aspects of child-rearing, even when deliberately presented by health educators, did not appear to be easily applied in the extended family setting, and long-term goals seemed inconsistent with the adolescent's behavior.

An NADP/thioredoxin system, consisting of NADPH, NADP-thioredoxin reductase (NTR), and its thioredoxin, thioredoxin h, has been previously described for heterotrophic plant tissues, i.e., wheat seeds and cultured carrot cells. Until now there was no evidence for this system in green leaves. Here, we report the identification of protein components of the NADP/thioredoxin system in leaves of several species. Thioredoxin h and NTR, which were both recovered in the extrachloroplastic fraction, were purified to apparent homogeneity from spinach leaves. This represents the first time that NTR has been characterized from a plant source. Similar to that from bacterial and mammalian sources, spinach leaf NTR was a flavoprotein (Mr 68,000) composed of two subunits of identical molecular mass (Mr 33,000) that resembled Escherichia coli NTR immunologically. Spinach thioredoxin h existed in two forms (Mr of 13,500 and 12,000) and was highly specific for plant NTR. Thioredoxin h and NTR partially purified from spinach roots showed properties similar to their counterparts from leaves. Spinach cytosolic thioredoxin h differed from chloroplast thioredoxin m or f from the same source but was similar to thioredoxin h from wheat seed in immunological properties.

Cell-free preparations from the green alga, Chlorella pyrenoidosa, contained two forms of phosphofructokinase (PFK), designated PFK I and PFK II. This represents the first evidence for a second form of PFK in green algae. A pyrophosphate D-fructose-6-phosphate, 1-phosphotransferase (PFP) activity, that was unaffected by the regulatory metabolite, fructose-2,6-bisphosphate, co-purified with PFK II through several steps. The data suggest that Chlorella pyrenoidosa resembles higher plants in containing two forms of PFK, but differs in containing an atypical form of PFP.

Temperature-sensitive mutant G31 of vesicular stomatitis virus induces mouse neuroblastoma N-18 cells to fuse during infections that are nonpermissive for virus replication, but BHK-21 cells do not undergo the viral glycoprotein-mediated cell fusion. The viral glycoprotein was expressed at the cell surface of both N-18 and BHK-21 cells; therefore, the host cell specificity did not stem from an absence of the viral glycoprotein at the surface of BHK-21 cells. Cell fusion readily occurred between infected and uninfected N-18 cells in mixed cultures, demonstrating that the viral glycoprotein was interacting with an uninfected cell for the initial cell-cell interaction of the cell fusion. Mixing infected BHK-21 cells with uninfected N-18 cells resulted in cell fusion initiated by BHK-21 cell-synthesized viral glycoprotein, but 88% of the nuclei in polykaryocytes were N-18 nuclei. The N-18 cell fusion specificity was readily apparent when infected N-18 cells were mixed with uninfected BHK-21 cells; 98% of the nuclei in polykaryocytes were N-18 nuclei. Similar results also were obtained with mixed cultures of N-18 cells and primary astroglial cells. Thus, the viral glycoprotein synthesized in any of the cell types could initiate cell fusion, but the properties of plasma membranes of neuroblastoma cells appeared to be much more suitable for cell-cell fusion.