Immediate-early, early, and late RNAs in bovine herpesvirus-4-infected cells. We have begun to identify immediate-early (IE), early (E), and late (L) genes of BHV-4 by analysis of cytoplasmic polyadenylated RNA transcribed from the BHV-4 genome over the course of infection and in the presence of cycloheximide or phosphonoacetic acid. Labeled cDNA prepared from RNA isolated at different times was hybridized with Southern blots of viral DNA to show which regions of the genome are transcribed at different times. In a second series of experiments, radiolabeled cloned restriction fragments representing the entire BHV-4 genome were hybridized separately to RNA on Northern blots to determine the number and sizes of transcripts at different times. As expected, with increasing time after infection, more portions of the BHV-4 genome are transcribed and a larger number and a greater abundance of viral transcripts are present. RNA transcribed from terminal repeats was not detectable at any time. However, similarity in size of RNA transcribed from opposite ends of the unique region of the genome late in infection suggests that RNA is transcribed over the fused ends of the genome, and terminal repeats are removed during RNA processing. Identification of IE, E, and L transcripts by this analysis lays the foundation for further study of specific BHV-4 transcripts and genes.


Membrane alterations linked to early interactions of HIV with the cell surface. Ultrastructural studies suggest that cell surface alterations occur early during the course of HIV-1 infection of CD4+T-lymphoblastoid cells. Attachment and penetration of HIV resulted in formation of membrane discontinuities and pores and "ballooning." Distention of the endoplasmic reticulum occurred in some cells within the first hour after HIV infection, and this correlated with the numbers of virions bound at the cell surface. These results suggest that HIV virion components may directly damage the cell membrane.


Somatostatin inhibits the norepinephrine-activated calcium channels in rMTC 6-23 cells: possible involvement of a pertussis toxin-sensitive G-protein. The mechanisms by which somatostatin inhibits hormone release are complex and involve, among other things, reduction of both intracellular cAMP and intracellular calcium. We studied the influence of the long-acting somatostatin analogue octreotide on norepinephrine (NE)-induced changes in intracellular calcium ([Ca2+]i) in fura-2 loaded single cells of a rat medullary carcinoma cell line, rMTC 6-23. Increases in the extracellular calcium concentration ([Ca2+]e) induced a sudden rise in [Ca2+]i which could be blocked by EGTA or the calcium channel blocker verapamil. NE evoked a similar increase in [Ca2+]i, which also could be blocked by the addition of EGTA or verapamil. Octreotide prevented or reversed the NE-induced increase in [Ca2+]i. Pretreatment of the cells with pertussis toxin abolished the inhibitory effect of octreotide. Thus we conclude that the NE-induced rise in [Ca2+]i is due to an influx of [Ca2+]e, most probably through voltage-dependent calcium channels. Octreotide inhibits the NE-stimulated rise in [Ca2+]i by a pertussis toxin-sensitive G-protein, most probably through a direct effect on NE-activated calcium channels.


Sebaceous gland and sweat gland carcinomas of the skin. Clinicopathological study and significance of c-erbB-2 oncoprotein expression. Thirteen sebaceous gland carcinomas and 10 sweat gland carcinomas were examined to elucidate any important histological parameters influencing their prognosis, and the relationship between immunohistochemical expressions of c-erbB-2 oncoprotein and survival of the patients was analyzed. Sebaceous gland carcinomas with vacuolated cytoplasm in more than 50% of whole tumor area, with necrosis, and without lymphoid cell infiltration in tumor nests and stroma had a higher incidence of tumor recurrence and tumor-related death than tumors with vacuolated cytoplasm in 50% or less of whole tumor area (p < 0.01), without necrosis, and with lymphoid cell infiltration in tumor nest and stroma (p < 0.05). Sweat gland carcinomas of all cases with fatal outcomes demonstrated tubular differentiation in 20% or less of whole tumor area, lymphatic permeation and desmoplastic reaction. Three sebaceous gland carcinomas and three sweat gland carcinomas were positive for c-erbB-2 oncoprotein. Two of three sebaceous gland carcinomas, and all three sweat gland carcinomas developed tumor recurrence and ended in tumor-related deaths. Sweat gland carcinomas with c-erbB-2 expression had significantly shorter survival than those with negative immunostain (p < 0.01). Cytoplasmic appearance, tumor necrosis, and lymphoid cell infiltration in tumor nests and stroma of sebaceous gland carcinoma, and tubular differentiation, lymphatic permeation, and growth patterns of sweat gland carcinoma are considered to closely correlate to the prognosis. Immunohistochemically detected c-erbB-2 oncoprotein may be an indicator of bad prognosis.


Expression of growth factors in a pancreatic islet regeneration model. In this model of pancreatic regeneration, the initial trophic effect of cellophane wrapping is upon the ductular-epithelium, as shown by autoradiographic analysis following a single pulse of 3H-TdR. The uptake of 3H-TdR by ductular cells is maximal approximately 4 weeks before the peak uptake of the label by islet cells. Six weeks after the pulse, most of the label is contained in differentiating islet cells, not ductular cells. This phenomenon suggests that wrapping of the pancreas induces "progenitor" cells to differentiate along the line of endocrine cells. The essentially negative observations on REG gene expression in the regenerating hamster pancreas indicate that the message for this gene is either not involved in the regeneration process or that it is sufficiently different from the rat gene (a rat control probe was used) that it precludes detection by our methodology. It might be necessary therefore, to generate a hamster REG specific probe in order to further study this issue. In conclusion, the established model of cellophane wrapping of the pancreas provides a useful tool whereby the induced growth of beta-cells can be observed. The sequence appears to be initial stimulation of ductal cells followed by islet differentiation. Of the candidate genes, it appears that reg does not participate in the process. We will however, examine other candidate genes to elucidate the mechanism whereby celophane wrapping induces growth of beta-cells.


Helicobacter pylori and peptic ulcers in rheumatoid arthritis patients receiving gold, sulfasalazine, and nonsteroidal anti-inflammatory drugs. The conflicting reports on gold and Helicobacter pylori could be related to the use of serological tests of unproven value in NSAID patients, and to the lack of the appropriate control groups. More important is the fact that the endoscopic consequences of the possible effect of gold on H. pylori have not been investigated. We therefore decided to assess the prevalence of H. pylori and peptic ulcers in rheumatoid patients being treated with gold sodium thiomalate (GST) plus NSAID, sulfasalazine plus NSAID, or NSAID only. Eighty-five patients receiving treatment for at least 6 months were endoscoped, and H. pylori was studied in gastric antral biopsies by both culture and histology. Endoscopic abnormalities were classified into ulcers (measuring 5 mm in diameter or more) and erosions (smaller lesions). H. pylori (and ulcers) were found in 17 (12 ulcers) of 31 patients on NSAID only and 21 (9 ulcers) of 27 patients on sulfasalazine plus NSAID, compared with nine (3 ulcers) of 27 patients receiving GST plus NSAID, p < 0.05, analysis of variance. Patients treated with GST and NSAID had the lowest prevalence of detectable H. pylori. This could explain the apparent reduction in the prevalence of peptic ulcers in this group and, if confirmed in larger randomized studies, might have therapeutic implications.


Vector competence of California mosquitoes for California encephalitis and California encephalitis-like viruses. Mosquitoes collected from coastal, inland valley, and alpine locations in California were evaluated for their experimental vector competence for two viruses in the California serogroup (Bunyaviridae:Bunyavirus). Aedes squamiger, a coastal salt marsh mosquito, was an efficient vector of a California encephalitis (CE)-like virus isolated from its habitat (89% of the pledget-fed females became infected and 61% transmitted virus). Aedes dorsalis, a coastal mosquito, and Ae. melanimon, an inland valley mosquito, were competent vectors of prototype CE virus (98% and 100% of the pledget-fed females became infected and 56% and 30%, respectively, transmitted virus). Aedes squamiger and Ae. dorsalis transmitted both viruses vertically to one or more of 20 of their progeny. Culiseta inornata was susceptible to infection with both viruses, but 5% or less transmitted virus perorally. Alpine mosquitoes, Ae. cataphylla, Ae. increpitus, and Ae. tahoensis, became infected with both CE and CE-like viruses, but 3% or less transmitted virus. All species of mosquitoes were more efficient vectors of both viruses following intrathoracic inoculation than following pledget feeding, suggesting the presence of mesenteronal barriers.


Linkage and association studies with C8A and C8B RFLPs on chromosome 1. Linkage relations for the C8A and C8B BamHI RFLPs have been investigated. A peak lod score of 4.52 at recombination fraction zero was obtained between the two C8 genes. Combined with our previously obtained linkage data (Rogde et al. 1986) the maximum lod score is 7.53 at recombination fraction zero. The compiled C8-PGM1 linkage data from this and the previous study gave a maximum lod score of 22.02 at recombination fraction 0.11 (0.07-0.16) with no sex difference. A chromosome 1p reference marker, D1S57, has been applied in this linkage study. A maximum lod score of 5.06 between the C8 cluster and D1S57 at theta = 0.18 (0.11-0.28) was recorded. The linkage analyses and triply informative families gave evidence that the C8 loci are situated about halfway between PGM1 and D1S57 on the short arm of chromosome 1. There was no evidence of allelic association between the C8A and C8B BamHI RFLPs in 62 unrelated haplotypes.


Structure and chromosomal localization of the human 2',3'-cyclic nucleotide 3'-phosphodiesterase gene. Human brain cDNA clones for the myelin associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) have been isolated and sequenced. The only 5' untranslated region (UTR) sequence found was that of a human CNPII mRNA, with no direct evidence for a CNPI mRNA. Human CNPase cDNAs were used to isolate genomic clones containing the human CNPase gene which is 9 kb long. Four exons were identified, separated by three introns, and the sequence of each exon and intron/exon boundary has been established. The polymerase chain reaction (PCR) was used to detect the presence of the human CNPase gene in DNA from a panel of rodent/human somatic cell hybrids. By this means the human CNPase gene was mapped to chromosome 17. In situ hybridization of a human CNPase genomic clone to metaphase chromosomes further localized this gene to chromosomal band 17q21.


Cortical biopsy in Alzheimer's disease: diagnostic accuracy and neurochemical, neuropathological, and cognitive correlations. Intraventricular Bethanecol Study Group. Neurochemical assessments were performed on biopsy samples taken from the right frontal lobe of patients diagnosed with Alzheimer's disease (AD), before the implantation of a ventricular catheter and pump assembly for the infusion of bethanechol chloride as an experimental therapy. The pathologically diagnosed patients with AD (n = 35; mean age, 67 +/- 1.5 yr) were compared with a group of samples from normal age-equivalent autopsied controls (n = 22; mean age, 68 +/- 2 yr) and autopsied AD brains (n = 11; mean age, 73 +/- 2 yr). Samples were assayed for choline acetyltransferase (ChAT), acetylcholinesterase, binding to [3H]quinuclidinyl benzilate as an index of total muscarinic cholinergic binding, and [3H]pirenzepine binding as an index of M1 cholinergic receptor subtype binding. Mean levels of ChAT activity were decreased in the biopsied patients to 36% of age-matched autopsied controls. The loss of ChAT activity correlated significantly with the Mini-Mental State Examination, an index of global cognitive function. Mean ChAT activity in autopsied AD cortex was further decreased compared with controls, indicating continuous decline through the course of the disease. Acetylcholinesterase followed a similar, less dramatic decline. No differences were found in [3H]quinuclidinyl benzilate binding or [3H]pirenzepine binding between biopsied and autopsied controls. Neuritic plaque counts did not correlate with either the Mini-Mental State Examination or ChAT activity in the biopsy specimens.(ABSTRACT TRUNCATED AT 250 WORDS)


Application of a monoclonal antibody-based antigen detection enzyme-linked immunosorbent assay (antigen ELISA) for field diagnosis of bovine trypanosomiasis at Nguruman, Kenya. A monoclonal antibody-based, enzyme immunoassay (antigen ELISA) for the detection of species-specific invariant antigens of Trypanosoma congolense, T. vivax or T. brucei in the serum of infected animals was evaluated as a means of diagnosis using bovine field sera from a trypanosomiasis endemic area, Nguruman, Kenya. Circulating trypanosome antigens were detected in 126 (96.2%) of 131 serum samples from animals with parasitologically confirmed diagnosis: 74.8% were positive for antigens of two or three trypanosome species, while 21.4% tested positive for one trypanosome species. When 70 sera from animals (at Nguruman), which had tested negative for trypanosomes by the buffy coat technique, were tested, 35 (50.0%) of them were shown to be antigen-ELISA positive: 24 (34.3%) showing infection with a single species and 11 (15.7%) with mixed infections. The predominant trypanosome species diagnosed in the two herds by antigen ELISA was T. vivax, which was detected in 133 (82.6%) of the 161 sera that tested positive for antigens, followed by T. congolense in 122 (75.8%) sera, with 109 (67.7%) showing evidence of mixed infections with two or three trypanosome species. In single infections, T. vivax exceeded T. congolense by a ratio of 2:1, with T. brucei accounting for less than 1.0%. Evidence for the specificity of the test was provided by analysis of field sera from 100 cattle, from a trypanosomiasis-free area, infected with other haemoparasites (anaplasmosis, babesiosis and theileriosis), which all tested negative in the assay.


Regional cerebral blood flow in monozygotic twins discordant and concordant for schizophrenia. We addressed several questions regarding hypofunction of the prefrontal cortex ("hypofrontality") in schizophrenia by measuring regional cerebral blood flow during three different cognitive conditions in monozygotic twins who were discordant or concordant for schizophrenia or who were both normal. These questions included the prevalence of hypofrontality, the importance of genetic predisposition, and the role of long-term neuroleptic treatment. Significant differences between affected and unaffected discordant twins were found only during a task linked to the prefrontal cortex, the Wisconsin Card Sorting Test. During this condition, all of the twins with schizophrenia were hypofrontal compared with their unaffected co-twins, suggesting that, if appropriate cognitive conditions and control groups are used, hypofrontality can be demonstrated in the majority of, if not all, patients with schizophrenia. When unaffected co-twins of patients with schizophrenia were compared with twins who were both normal, no differences were observed, suggesting that nongenetic factors are important in the cause of the prefrontal physiologic deficit that appears to characterize schizophrenia. When concordant twins with a high- vs a low-dose lifetime history of neuroleptic treatment were compared, the twin receiving the higher dose was more hyperfrontal in six of eight pairs, suggesting that long-term neuroleptic treatment does not play a major role in hypofrontality.


Frontostriatal disorder of cerebral metabolism in never-medicated schizophrenics. We scanned 18 patients with schizophrenia who had never received neuroleptic medication and 20 age- and sex-matched controls by positron emission tomography with 18-F-fluorodeoxyglucose (fludeoxyglucose F 18) as a tracer of glucose metabolism. Subjects performed the Continuous Performance Test during 18-F-fluorodeoxyglucose uptake. Scan results were converted to metabolic rates, and computer algorithms were used to identify cortical regions. Previous reports of relative hypofrontality in schizophrenia were confirmed, indicating that this finding is not an artifact of previous treatment. Significantly reduced ratios of inferior and medial frontal regions to occipital cortex were found, together with diminished metabolism in the basal ganglia. This suggests the presence of a combined frontostriatal dysfunction in schizophrenia.


Hypofrontality in neuroleptic-naive patients and in patients with chronic schizophrenia. Assessment with xenon 133 single-photon emission computed tomography and the Tower of London. The "hypofrontality hypothesis" has been supported by many neuroimaging studies, but not all, perhaps because of heterogeneity of samples. The present study examined three different samples that permitted assessment of a variety of confounders, such as effects of long-term treatment, chronicity of illness, and presenting phenomenology: (1) 13 neuroleptic-naive schizophrenic patients, (2) 23 nonnaive schizophrenic patients who had been relatively chronically ill but were medication free for at least 3 weeks, and (3) 15 healthy normal volunteers. Regional cerebral blood flow was measured using single-photon emission computed tomography with xenon 133 as the tracer. The control condition consisted of looking at undulating colored shapes on a video monitor, while the experimental task was the Tower of London. We observed the Tower of London to be a relatively specific stimulant of the left mesial frontal cortex (probably including parts of the cingulate gyrus) in healthy normal volunteers. Both the neuroleptic-naive and the nonnaive patients lacked this area of activation, as well as a related one in the right parietal cortex (representing the circuitry specifically activated by the Tower of London). Decreased activation occurred only in the patients with high scores for negative symptoms. These results suggest that hypofrontality is related to negative symptoms and is not a long-term effect of neuroleptic treatment or of chronicity of illness.


Negative symptoms and hypofrontality in chronic schizophrenia. Frontal lobe dysfunction is widely suspected to underlie negative symptoms of schizophrenia. This hypothesis is based largely on long-standing observations of the similarities between the effects of frontal lobe lesions and negative symptoms. However, there is little direct evidence specifically for such an association in schizophrenic patients. We measured the relationship between decreased relative prefrontal cortex glucose metabolism (hypofrontality) using positron emission tomography and evaluated the severity of negative symptoms in 20 chronic schizophrenics who underwent scanning while not receiving neuroleptic drugs. We found a close relationship between negative symptoms and prefrontal hypometabolism, particularly in the right dorsolateral convexity. This association was regionally specific. Furthermore, there was no evidence that this relationship was an artifact of age, cerebral atrophy, or severity of positive symptoms.


Schizophrenia, genetic retrenchment, and epidemiologic renaissance. The Sixth Biennial Winter Workshop on Schizophrenia, Badgastein, Austria, January 26-February 1, 1992. A distinctive feature of these workshops, in addition to those noted in the introductory overview, is the selection of a relatively isolated location for a 1-week period. This, together with a rich and varied program and an ethos of informality, encourages participants to discuss not only the work presented but also their unpublished work and their intuitions based on preliminary data and analyses. Such an interchange is of inestimable value to the schizophrenia research community. In scientific terms, a panel of concluding discussants (Drs Kendell, Torrey, and Waddington) were in some measure of agreement that genetics, particularly molecular genetics, appears to be experiencing a period of retrenchment, while epidemiology is experiencing something of a renaissance. Maternal influenza was a prominent theme, although the data were far from consistent. It was argued by Dr Wessely that risk for schizophrenia putatively attributable to maternal influenza might be 5% to 10% of all cases, indicating a modest effect. Eclectically, Dr Kendell believed the effect to be "real" but slight and fragile, it being sought against large aggregates that almost inevitably result in differing findings from differing countries or from different data bases within a given country. Gender differences were also among the more prominent themes, not just in an epidemiologic context but also in a variety of other studies. This points anew to disturbances in schizophrenia of factors that regulate, or are intimately associated with, sexual dimorphism in brain development. Abnormalities in cerebral asymmetry continue to pervade a variety of research findings and point further to neurodevelopmental anomalies.(ABSTRACT TRUNCATED AT 250 WORDS)


Sensory testing in human immunodeficiency virus type 1-infected men. HIV Neurobehavioral Research Center Group. Patients with acquired immunodeficiency syndrome frequently suffer peripheral neuropathy. We investigated its prevalence and relationship to clinical stage of human immunodeficiency virus (HIV) infection using quantitative sensory testing and nerve conduction testing. Vibratory threshold was determined in the right great toe and index finger of 179 men seropositive for HIV (28 with acquired immunodeficiency syndrome [AIDS] or AIDS-related complex [ARC], 151 asymptomatic) and 32 HIV-seronegative controls. None had clinical peripheral neuropathy. Abnormal threshold was control mean plus 2.5 SDs. In the toe, 10 (36%) of 28 subjects with AIDS or ARC had abnormal vibratory thresholds, compared with seven (5%) of 151 asymptomatic seropositive subjects and none of 32 controls. A subgroup of 168 seropositive subjects underwent nerve conduction testing. Abnormality rates were similar, but abnormalities of nerve conduction coincided with quantitative sensory testing abnormalities in only half the cases. Mean (+/-SD) vibratory threshold was significantly greater in subjects with AIDS or ARC (3.00 +/- 0.51 vibratory units) than in asymptomatic subjects (1.56 +/- 0.27 vibratory units) and controls (1.63 +/- 0.54 vibratory units). Finger abnormality rates did not differ, although subjects with AIDS or ARC had greater mean vibratory threshold. Subclinical peripheral neuropathy is thus related to stage of HIV infection and is present by quantitative sensory testing in 36% of patients with AIDS or ARC.


Restriction enzyme analysis of Norrie disease pedigrees. Carrier detection and prenatal diagnosis of Norrie disease (ND) were performed using the polymorphic L1.28, OTC, and 58-1 probes. L1.28 was polymorphic in 3 of the 5 ND families tested and informative in 2 families (40%). Probe 58-1 and OTC were informative in one of 3 families (33%) and in both of the 2 families (100%) tested, respectively. The major allele frequency was 73% in L1.28 (DXS7), 89% in 58-1 (DXS14), and 50% in OTC in our patient population. One of 5 families studies showed a recombination between probes (L1.28 and OTC) and the ND gene locus placing the ND locus proximal to L1.28 and OTC.


Escherichia coli gamma-glutamyltranspeptidase mutants deficient in processing to subunits. Arginyl residues 513 and 571 of Escherichia coli K-12 gamma-glutamyl-transpeptidase (EC 2.3.2.2) were substituted with alanyl and glycyl residues, respectively, by oligonucleotide-directed in vitro mutagenesis. Both mutants were devoid of the enzymatic activity. On Western blot analysis, we found that both mutants accumulated a gamma-glutamyltranspeptidase precursor which was not processed into large and small subunits in the periplasmic space of Escherichia coli.


Stable expression of natriuretic peptide receptors: effects of HS-142-1, a non-peptide ANP antagonist. We established clonal cell lines stably expressing each of two subtypes of membrane bound guanylate cyclases (GC-A and GC-B), which are known as natriuretic peptide receptors. Using these cell lines, we showed that GC-A is an ANP/BNP receptor, whereas GC-B is a specific receptor for CNP. Effects of HS-142-1, a novel non-peptide ANP antagonist, on GC-A and GC-B were examined by using these cells. In cells expressing either GC-A or GC-B, HS-142-1 inhibited cGMP production elicited by ANP or CNP with IC50 values of 1.8 micrograms/ml and 1.5 micrograms/ml, respectively, and also competitively blocked specific binding of the natriuretic peptides with IC50 values of 2.2 micrograms/ml and 3.3 micrograms/ml, respectively. These results indicate that HS-142-1 is a potent antagonist of CNP as well as ANP. We also showed that CNP suppressed the growth of cells expressing GC-B by 22% and that HS-142-1 blocked the antiproliferative action of CNP.


The MDR1 gene product, P-glycoprotein, mediates the transport of the cardiac glycoside, digoxin. Digoxin, a widely used cardiac glycoside with a low therapeutic index, is known to interact with a large and diverse group of co-administered drugs, frequently leading to toxic accumulation of the glycoside. Establishing the mechanism(s) of these interactions, therefore, has potential clinical significance. The present studies implicate P-glycoprotein, the MDR1 gene product overexpressed in multidrug resistant cells, as the apical membrane protein responsible for the renal secretion of digoxin and provide an explanation for the occurrence of digoxin toxicity in the presence of certain co-administered medications. Since digoxin is considered a prototype for endogenous digitalis-like glycosides, the results also allow for speculation that endogenous digitalis-like glycosides may be the natural substrates for P-gp.


Production of catecholamines in the human epidermis. Cell-free extracts from human full thickness skin (i.e., epidermis and dermis), suction blister roofs (i.e., epidermis) and from human keratinocytes express biopterin-dependent tyrosine hydroxylase a well as phenylethanolamine-N-methyl transferase, both representing key enzymes for the biosynthesis of epinephrine. These enzyme activities could not be detected in cell extracts from human melanocytes and human fibroblasts. Since keratinocytes in the human epidermis, and in cell cultures, express a high density of beta-2-adrenoceptors, and this signal transduction system regulates intracellular calcium homeostasis, it can be concluded that epinephrine production in the epidermis activates calcium transport via the beta-2-adrenoceptor system. Our results show for the first time that the human epidermis has the capacity to independently produce epinephrine.


Multidrug resistant HOB1 lymphoma cells express P-glycoprotein that does not play the major role in the development of drug resistance to adriamycin. Two cell lines resistant to 0.1 microM vincristine (VCR) and 2.0 microM adriamycin (ADR), respectively, (designated HOB1/VCR0.1 and HOB1/ADR2.0) were established from a human immunoblastic B lymphoma cell line. These cell lines showed the typical MDR phenotype with overexpression of P-glycoprotein and decreased [3H]VCR accumulation. The retention amounts of intracellular [3H]VCR in these two cell lines could be augmented by verapamil. However, in spite of the overproduction of P-glycoprotein, both HOB1/VCR1.0 and HOB1/ADR2.0 cells did not exhibit decreased accumulation of intracellular [14C]ADR. And the retention of [14C]ADR was not affected by verapamil. Our data support that P-glycoprotein is a drug transporter more important for the development of drug resistance to VCR than to ADR.


Effects of a new triazinoaminopiperidine derivative on adriamycin accumulation and retention in cells displaying P-glycoprotein-mediated multidrug resistance. A new triazinoaminopiperidine derivative, Servier 9788 (S9788), was investigated for its ability to increase Adriamycin (ADR) accumulation and retention in two rodent (P388/ADR and DC-3F/AD) and three human (KB-A1, K562/R and COLO 320DM) cell lines displaying the P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) phenotype. Depending on the cell line S9788 was shown to be two to five times more active and five to 15 times more potent than Verapamil (VRP) in increasing ADR accumulation in resistant cells. ADR retention in KB-A1 cells maintained in a concentration of 10 microM S9788 was twice that in VRP-treated cells, and similar to that measured in the untreated sensitive KB-3-1 cells. Although 5 microM S9788 and 50 microM VRP gave the same values of ADR uptake in KB-A1 cells, S9788 was shown to induce a greater ADR retention following cell wash and post-incubation in resistance modifier- and ADR-free medium. Taking into account that S9788 had no effects on ADR accumulation and retention in sensitive KB-3-1 cells, it can be suggested that S9788 inhibits specifically the P-gp dependent ADR efflux, and in a manner less reversible than that observed with VRP. Moreover, [3H]azidopine photolabeling of P-gp, in P388/ADR plasma membranes, was completely inhibited by 100 microM S9788. Although S9788, as VRP, had no effect on the cell cycle of P388 cells, 5 microM S9788 increased 700-fold the efficacy of ADR to block P388/ADR cells in the G2+M phase of the cell cycle. Together, these results show that the sensitization, by S9788, of cell lines resistant to ADR is mainly due to an increase in ADR accumulation and retention, leading to an increase in the number of resistant cells blocked in the G2+M phase.


Drug interactions in intestinal transport of folic acid and methotrexate. Further evidence for the heterogeneity of folate transport in the human small intestine. The effect of sulfasalazine and olsalazine on the transport of [3H]folic acid and of [3H]methotrexate (MTX) was investigated in organ-cultured endoscopic biopsy specimens of small intestinal mucosa from normal subjects. Biopsy specimens obtained from patients undergoing routine diagnostic upper gastrointestinal endoscopy were organ-cultured at pH 5.5 and the effect of these two drugs on the initial rate of uptake of the two folates was determined. Both drugs inhibited the transport of [3H]folic acid with similar Ki values (1.38 and 1.32 mM for sulfasalazine and olsalazine, respectively). However, the uptake of [3H]MTX was only partially inhibited by sulfasalazine and was unaffected by olsalazine. Sulfasalazine inhibited 26.2% of the total flux of MTX, in close agreement with the fraction of MTX flux that has been shown previously to be inhibited by folic acid. These data corroborate previous findings of heterogeneity of transport of MTX in the mucosa of the human small intestine.


Inhibition of aminopeptidases N, A and W. A re-evaluation of the actions of bestatin and inhibitors of angiotensin converting enzyme. The effects of a range of metallopeptidase inhibitors on the activities of the porcine kidney cell surface zinc aminopeptidases, aminopeptidase A (AP-A; EC 3.4.11.2), aminopeptidase N (AP-N; EC 3.4.11.7) and aminopeptidase W (AP-W; EC 3.4.11.16), have been directly compared. Amastatin and probestin were effective against all three aminopeptidases, with the concentration of inhibitor required to cause 50% inhibition (I50) in the low micromolar range (I50 = 1.5-20 microM), except for probestin with AP-N which displayed an I50 of 50 nM. Actinonin failed to inhibit significantly either AP-A or AP-W, and thus can be considered a relatively selective inhibitor (I50 = 2.0 microM) of AP-N. In contrast, bestatin was a relatively poor inhibitor of AP-N (I50 = 89 microM) and failed to inhibit AP-A, but was more potent towards AP-W (I50 = 7.9 microM). Thus, some of the observed chemotherapeutic actions of bestatin may be due to inhibition of cell-surface AP-W. A number of other metallopeptidase inhibitors, including inhibitors of endopeptidase-24.11 (EC 3.4.24.11) and membrane dipeptidase (EC 3.4.13.11), and the carboxylalkyl and phosphoryl inhibitors of angiotensin converting enzyme (EC 3.4.15.1) failed to inhibit significantly AP-A, AP-N or AP-W. However, AP-W was inhibited with I50 values in the micromolar range by the sulphydryl converting enzyme inhibitors rentiapril (I50 = 1.6 microM), zofenoprilat (I50 = 7.0 microM) and YS 980 (I50 = 17.7 microM). Neither AP-A nor AP-N were affected by these sulphydryl compounds. Inhibition of AP-W may account for some of the side effects noted with the clinical use of the sulphydryl converting enzyme inhibitors. The availability of compounds which are totally selective for AP-W over any of the other mammalian cell surface zinc aminopeptidases may aid in identifying endogenous substrates, and thus physiological or pathophysiological role(s) of AP-W.


Characterization of adriamycin-resistant and radiation-sensitive Chinese hamster cell lines. A series of cell lines derived from Chinese hamster V79 cells by selection in increasing concentrations of Adriamycin (ADRM) was developed to study the mechanisms of drug resistance and its relationship to radiation response. Survival studies revealed that selection in increasingly higher concentrations of ADRM positively correlated with increased cellular drug resistance. Increased cellular resistance correlated positively with amplification of the hamster multidrug-resistance gene (pgp 1) as detected with dot blot analysis using the pCHP1 probe. Southern blot analysis of restriction endonuclease digested DNA (Eco RI, Hind III, Pst I, or Bam HI) showed that (1) some fragments were preferentially amplified compared to others in the ADRM-resistant lines; and (2) no major gene rearrangement appeared to have occurred during the selection for greater ADRM resistance. Levels of pgp 1 gene expression assayed with dot blot and Northern analysis showed a parallel increase of mRNA with gene amplification and increased ADRM resistance. The amounts of the pgp 1 gene product, P-glycoprotein (P-gp), in the cell membrane of the ADRM-resistant cells correlated with the amount of gene amplification/expression. However, levels of P-gp only correlated with degree of drug resistance as measured by cell survival in earlier selection stages (77A and LZ-3). In later selection stages (LZ-8 and LZ-24), higher levels of ADRM resistance were achieved but levels of P-gp did not increase beyond approximately 20% of plasma membrane proteins. These results suggest that (1) the LZ cell plasma membrane may have a physical limit as to the amount of P-gp it can accommodate and/or there is a cellular mechanism for regulating the amount of P-gp in the plasma membrane, and (2) additional resistance mechanisms are present in LZ-8 and LZ-24 cells. Microscopic observations of intracellular drug distribution in these cell lines revealed that (1) ADRM appeared to be sequestered in cytoplasmic vesicles, and (2) the amount of sequestration (number of vesicles) exhibited correlated with the degree of drug resistance attained by the cell lines. These results suggest that drug sequestration is another mechanism of resistance in LZ cells in addition to P-gp-mediated drug efflux.


An enhanced ability for transforming adriamycin into a noncytotoxic form in a multidrug-resistant cell line (LZ-8). Multidrug-resistant LZ-8 cells are 9000-fold more resistant to Adriamycin (ADRM) exposure than wild-type V79 cells. To understand more about the mechanisms producing such high level resistance, we tested whether LZ-8 cells inactivate ADRM toxicity to a greater extent than wild-type V79 cells. ADRM was recovered from (1) culture media of wild-type V79 and ADRM-resistant LZ-8 cells; (2) V79 and LZ-8 cells; and (3) LZ-8 cell plasma membrane, and the cytotoxicity was determined by treating V79 cells for 1 hr with a known concentration of the recovered ADRM. ADRM obtained from LZ-8 cells or its culture medium exhibited less cytotoxicity than that recovered from V79 cells or its culture medium. ADRM extracted from LZ-8 cell plasma membrane was noncytotoxic. HPLC analysis revealed that the extracted ADRM was structurally changed compared to stock ADRM. The retention time in the column was 7 min for stock ADRM, and 23 min for the recovered ADRM. Thus, LZ-8 cells have an increased ability to transform ADRM into a noncytotoxic form compared to wild-type V79 cells. This transformation involves structural conversion into a previously unidentified ADRM metabolite. The greatly increased survival of LZ-8 cells compared to V79 cells after ADRM treatment is due to at least two mechanisms: (1) an enhanced ability to inactivate the cytotoxicity of ADRM, and (2) increased drug efflux resulting from the amplification and overexpression of the pgp 1 gene in these cells. Our results suggest the possibility that P-glycoprotein participates in drug binding/inactivation in addition to serving as a drug efflux pump.


Alcohol-induced macrocytosis and blood pressure. The effect of alcohol on blood pressure was studied prospectively in consecutive general practice patients with macrocytosis (MCV greater than or equal to 100 fl). The patients were separated into misuser and non-misuser groups on the basis of the Malmö modified Michigan Alcoholism Screening Test. There was no significant difference in the prevalence of antihypertensive medication between the misuser and non-misuser groups. When patients using antihypertensive medication were excluded and the groups were age-adjusted, male misusers (n = 95) compared to control patients (n = 22) had significantly higher diastolic (88 mmHg and 81 mmHg, respectively, P = 0.001) and systolic (146 mmHg and 137 mmHg, respectively, P less than 0.001) blood pressure values. Female misusers (n = 24), as compared to female non-misusers (n = 59) had significantly higher diastolic (83 mmHg and 82 mmHg, respectively, P = 0.04) but not systolic blood pressure values. Thus, alcohol seems to have a pressor effect predominantly among men. As 72% of men with macrocytosis were alcohol misusers and 41% of them either had elevated systolic or diastolic blood pressure, all patients with macrocytosis should be asked about their alcohol consumption and at least the males should have blood pressure measured.


The A14 position of insulin tolerates considerable structural alterations with modest effects on the biological behavior of the hormone. As part of our aim to investigate the contribution of the tyrosine residue found in the 14 position of the A-chain to the biological activity of insulin, we have synthesized six insulin analogues in which the A14 Tyr has been substituted by a variety of amino acid residues. We have selected three hydrophilic and charged residues--glutamic acid, histidine, and lysine--as well as three hydrophobic residues--cycloleucine, cyclohexylalanine, and naphthyl-(1)-alanine--to replace the A14 Tyr. All six analogues exhibit full agonist activity, reaching the same maximum stimulation of lipogenesis as is achieved with porcine insulin. The potency for five of the six analogues, [A14 Glu]-, [A14 His]-, [A14 Lys]-, [A14 cycloleucine]-, and [A14 naphthyl-(1)-alanine]-insulins in receptor binding assays ranges from 40-71% and in stimulation of lipogenesis ranges from 35-120% relative to porcine insulin. In contrast, the potency of the sixth analogue, [A14 cyclohexylalanine]insulin, in both types of assays is less than 1% of the natural hormone. The retention time on reversed-phase high-performance liquid chromatography for the first five analogues is similar to that of bovine insulin, whereas for the sixth analogue, [A14 cyclohexylalanine]insulin, it is approximately 11 min longer than that of the natural hormone. This suggests a profound change in conformation of the latter analogue. Apparently, the A14 position of insulin can tolerate a wide latitude of structural alterations without substantial decrease in potency. This suggests that the A14 position does not participate directly in insulin receptor interaction. Only when a substitution which has the potential to disrupt the conformation of the molecule is made at this position, is the affinity for the receptor, and hence the biological potency, greatly reduced.


Immunocytochemical localization of cyclin/proliferating cell nuclear antigen (PCNA) in fertilized eggs of mice. Immunolocalization of cyclin/PCNA (proliferating cell nuclear antigen) was performed with monoclonal antibody using immunogold methods on ultrathin cryosections of fertilized mouse eggs. Immunolabeling in pronuclei was checked 20, 22, 24 and 26 h after HCG injection. A relation between onset of pronuclei migration (early S-phase) and appearance of colloidal particle clusters was found. Afterwards, (mid S-phase) the increase of labelling and the localization of cyclin/PCNA were found throughout the pronuclei, except in the nucleolar bodies. Lower labelling appeared at the time of close reciprocal pronuclei contact (late S-phase). It is concluded that bulk and distribution of cyclin/PCNA in pronuclei is closely related to the progression of first interphase after fertilization.


Plasma and tissue levels of digestive regulatory peptides during postnatal development and weaning in the calf. Changes in the concentrations of cholecystokinin, gastric inhibitory peptide, gastrin, motilin, pancreatic polypeptide, secretin, somatostatin, and vasoactive intestinal peptide in calf plasma and antral, duodenal and/or pancreatic tissues were assessed by radioimmunoassay during postnatal development and after weaning in 50 male Holstein-Friesian calves (randomly distributed into 10 groups of 5 animals each). The calves in the first group were killed at birth while those in 6 other groups were colostrum-fed for 2 days and then milk-fed until 7, 28, 56, 70 or 119 days of age. Those in the remaining 3 groups were given the same diets until day 28, were then weaned between day 29-56, and slaughtered on days 56, 70 or 119. In milk-fed animals, changes in plasma and tissue concentrations of almost all digestive regulatory peptides were observed during the 1st month of postnatal life, especially at day 2. Weaning was accompanied by variations in the plasma concentrations of somatostatin, secretin, gastrin, pancreatic polypeptide and gastric inhibitory peptide but not by any apparent change in peptide tissue concentrations (except VIP in the duodenum). Thus, the variations in tissue concentrations are primarily age-related, while plasma concentrations were modified by age and weaning.


Studies on the activation by dolichol-P-mannose of the biosynthesis of GlcNAc-P-P-dolichol and the topography of the GlcNAc-transferases concerned with the synthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol: a review. A review is presented of research carried out in this laboratory on two aspects of the dolichol pathway that may have regulatory influences on these events. (i) The validity of the phenomenon of the activation of the biosynthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol by dolichol-P-mannose is supported by experiments carried out on the Thy-1-negative mouse lymphoma cell. While this cell cannot synthesize the activating compound, this capacity was retained and revealed upon the addition of exogenous dolichol-P-mannose. (ii) The topographical orientation of the GlcNAc-transferases that catalyze the biosynthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol was investigated in microsomes from the liver of the embryonic chick using dolichol phosphate liposomes as an exogenous substrate. The formation of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol was inhibited by trypsinization under conditions where the native orientation of the microsome was maintained, as indicated by the latency of mannose-6-phosphatase. Both GlcNAc-lipids were detected on free liposomes after incubation with intact microsomes, and in the same proportions as found on the microsome. From these and other studies, evidence was obtained indicating the cytoplasmic orientation of the GlcNAc-transferases that catalyze the synthesis of the first two intermediates of the dolichol pathway.


Treatment of traumatic dental injuries in children. The major emphasis of this review should rest on articles written within the past 12 months. Many of these papers comprise comprehensive surveys of treatment of various aspects of dental trauma in the primary and permanent dentition, as well as epidemiological studies. There have also been some very good experimental studies which have attempted to standardize luxation injuries as well as tooth replantation in order to study pulpal and periodontal healing in reproducible animal models. However, it can be seen from the reference list that one year's production in dental traumatology would not suffice to cover the scope of the treatment needs nor to adequately orient the reader on the progress made with respect to our present knowledge of wound healing following injury and the most recent developments in the restoration of the traumatized dentition. To present an overall view of the philosophy which has evolved concerning wound healing in the dental pulp and periodontium following injury as well as innovations in the treatment of acute dental trauma, this review must of necessity delve back into the mid-80's and probe forward into publications which are on the way in 1991. In 1984, in Dallas, Texas, the American Association of Endodontists' Endowment and Memorial Foundation convened the first International Conference on Oral Trauma. In 1989, in Stockholm, Sweden, this was followed up with the second International Conference on Dental Trauma. The one-day program summarized the state of the art of dental traumatology with respect to various aspects of epidemiology, diagnosis, treatment and prevention as well as the biological principles which form a basis for our present knowledge about healing and healing complications in the pulp and periodontium after traumatic dental injuries. The proceedings from this conference have been compiled in a newly published volume. Where applicable, these presentations have been cited as current reviews. It could also be mentioned that at the conclusion of the second conference, in recognition of the need to promote research and propagate knowledge in the area of dental traumatology, the International Association of Dental Trauma (IADT) was founded. The third International Conference on Dental Trauma was convened in Copenhagen, Denmark, June 1991. The major theme was restoration of the traumatized dentition. Proceedings from this conference are to be published. However, new information from that program is also cited in the following where applicable. Finally, the second Charlotte Conference on Pathobiology of the Dentin/Pulp Complex was held in Charlotte, North Carolina in May 1991.(ABSTRACT TRUNCATED AT 400 WORDS)


Inadequate internal mammary artery graft as a cause of postoperative ischemia: incidence, diagnosis and management. Inadequate left internal mammary artery (LIMA) graft to the left anterior descending artery (LAD) was encountered in 10 of 3,076 patients between 1984 and July 1990. The mean number of bypass grafts was 2.9 per patient. All patients with inadequate LIMA grafts were stable preoperatively with normal to moderately reduced left ventricular function. No technical difficulties were encountered during surgery. All patients were weaned off cardiopulmonary bypass with minimal or no inotropic support. Each patient developed myocardial ischemia of the LAD territory and/or circulatory collapse or recurrent ventricular dysrhythmia during the first 24 h postoperatively. Six patients, who were immediately re-operated on and had an additional saphenous graft to the LAD, recovered with no infarction and good functional results. Four patients, who were medically treated, developed myocardial infarction. In cases of refractory circulatory collapse and/or ventricular dysrhythmia, inadequate LIMA flow should be suspected. We recommend urgent re-operation with additional saphenous vein graft to the LAD.


Treatment of a dental phobic with pronounced aversion to rubber gloves. This case illustrates where excessive dental stress on the swallowing reflex caused retching then nausea and eventually dental phobia. Swallowing relaxation enabled normal variable function to be quickly restored, which allowed the phobia to be brought under control.


Heads and tales: recent advances in craniofacial development. In the last few years, our understanding of the complex series of events governing craniofacial morphogenesis has significantly increased. A variety of experimental approaches, such as cell lineage marking, monoclonal antibodies, organ culture and recombinant DNA techniques, have been fundamental to this progress. The neural crest contribution to the head and face of the embryo has been mapped, the significance and mechanisms of epithelial-mesenchymal interactions are better understood, and a number of regulatory molecules, which are likely to be causally involved in mediating the morphogenesis of the different craniofacial regions, have been identified. Although the information presently available on the molecules involved is far from complete, and the experimental strategies need further refinement, the existence of specific combinatorial gene-codes (ie the homeobox-containing gene code) has become evident. These findings are proving invaluable to our understanding of the mechanisms underlying craniofacial development, and of certain syndromes affecting craniofacial structures in humans.


Inflammation of the subacromial bursa in chronic shoulder pain. Subacromial bursal tissue was studied in 12 patients operated on for painful (10 patients with constant pain and 2 patients with pain on motion) rotator cuff tendinitis/impingement syndrome. The Neer acromioplasty technique was used. Six patients had moderate inflammatory changes and one had a slight inflammation. In three of the five remaining patients, the subacromial bursa did not show any signs of inflammatory involvement, but patients experienced pain at rest and at night, reflecting clinical inflammation in tissues other than the bursa. The two patients with pain only on strain did not show inflammation of the bursa. Immunohistochemical typing of the bursal tissue disclosed a typical chronic mononuclear cell infiltrate consisting mainly of CD2-positive T lymphocytes (50-80% of all inflammatory cells), accompanied by less frequent CD11b (C3bi receptor)-positive monocyte/macrophages (10-40%). The relative paucity of plasmablasts/plasma cells expressing PCA-1 suggests this to be an inflammatory rather than an immune response. Active involvement of some of the local cells is suggested to be the source of algogenic and hyperalgesic substances contributing to pain in chronic shoulder pain syndromes.


c-erbB2 amplification and overexpression in human tumors. There is no evidence for activation of c-erbB2 by mutation in human cancer. Gene rearrangements are observed at low frequency, but there are a proportion of human cancers that are associated with c-erbB2 gene amplification and membrane protein overexpression. The human cancers so affected are adenocarcinomas of the breast, ovary, stomach, and bladder, with up to 20% of primary lesions exhibiting either increased gene copy number and/or excess membrane staining. The c-erbB2 protein on these tumors could be used as a therapeutic target, as in monoclonal antibody targetted therapy already being assessed in c-erbB2 positive breast cancer. Other possible therapeutic strategies include the development of tyrosine kinase inhibitors or ligand antagonists.


Tyrosine kinase receptor--nuclear protooncogene interactions in breast cancer. In summary, evidence is beginning to accumulate in support of a major role for tyrosine kinase receptors (and their activating growth factors) and steroid hormones and their receptors in normal development and differentiation of the mammary gland. A point of intersection of their mechanisms of action in growth control appears to be the induction of nuclear protooncogenes such as c-myc. When c-myc is amplified, as it is in many breast cancers, EGF and FGF receptor tyrosine kinase action becomes transforming, not simply mitogenic. A source of the transforming factors could be either stromal or epithelial. This mechanism could function early in the progression of breast cancer. c-erbB-2 and EGF receptor overexpression and amplification, when they occur, appear to render tumors even more malignant and of especially poor prognosis. These mechanisms could function late in the progression of breast cancer. Transgenic mouse studies have begun to echo these themes. They have established that a growth factor (TGF-alpha) and its receptor (EGF receptor), which appear to be important in normal mouse and human proliferation and gland development, and a protooncogene (c-myc), commonly amplified and overexpressed in human and mouse breast cancer, can each contribute to mammary carcinogenesis. The mechanisms of the two are likely to be distinct. myc is likely to be acting as a tumor initiator in combination with normal proliferative factors, whereas TGF-alpha is likely to be acting as a hyperproliferative (promotional) factor in combination with a normal background of mutational events. The role of unmutated but amplified erbB-2 in the transgenic mouse is not yet known.


Suppressor genes in breast cancer: an overview. It is apparent that multiple genetic events occur in the development and progression of breast cancer. From the limited data available, no consistent temporal pattern of mutational events is required. This conclusion is consistent with data in colorectal carcinoma, where the number of mutational events, and not the order, appears to be relevant. Several authors have questioned whether the multiple mutational events occur independently or whether significant associations were evident. Cropp et al. postulated that two sets of mutational events occurred simultaneously in a higher degree of breast tumors than expected based on chance: Set 1 consisted of deletions on 11p, 17p, 18q, and int-2 and myc amplifications; set 2 consisted of 17q, 1p, and 3p deletions. Sato et al., analyzing another tumor cohort for simultaneous mutations, noted a correlation of 17p and 16q deletions, 13q and 17p deletions, and 17p deletion with erbB-2 amplification. Clearly, concordant data on this issue will require the use of large breast tumor cohorts for a comprehensive set of probes. The reasons why mutations to specific genes on different chromosomes tend to occur coordinately is unknown, but may involve common flanking and/or intron sequences at high risk for certain types of mutational events. Another interesting question is the degree to which alterations, but not homozygous inactivation, of suppressor genes occur and its phenotypic consequences. In this chapter, evidence was presented for the amplification of a DCC allele in breast cancer and for variable RB protein expression in breast tumors as a consequence of allelic deletion. For many of the metastasis suppressor genes, a simple reduction in their expression, or an alteration in their expression over the normal cellular regulatory controls, may be sufficient to fuel the metastatic process. The data suggest a more complex regulation of the cancer phenotype by suppressor genes than by recessive inactivation alone. Why do many sporadic cancers, including breast cancer, appear to require alterations to multiple suppressor genes, as compared to diseases such as retinoblastoma, where a single suppressor gene appears to control the cancer phenotype? The answer to this question is unknown, but most theories are based on the hypothesis that suppressor genes act to control cellular responses to either other cells or signals in the microenvironment. In retinoblastoma all cells can carry a germ-line mutation. Cells carrying the RB mutation can interact with both the embryonic and differentiated microenvironments; the specific interaction of mutated cells with the embryonic retinal microenvironment may trigger the onset of retinoblastoma.(ABSTRACT TRUNCATED AT 400 WORDS)


The role of the retinoblastoma gene in breast cancer development. The observation that the human retinoblastoma gene is inactivated in about 20% of breast carcinomas indicates that it may be important in the development of these tumors. The fact that the loss of RB1 expression correlates with the progression of the disease, and especially with the inability of the cells to differentiate, is consistent with the clinical observation that retinoblastoma does not occur in children in whom the target cells have already fully differentiated. This suggests that the normal function of RB1 is to promote differentiation. It is possible that the loss of the ability of a cell to differentiate contributes to its ability to grow in a foreign environment (metastasis), but this hypothesis remains to be tested. Our observation that the overexpression of RB1 suppresses the growth of these tumor cells in vitro is consistent with this hypotheses.


Stereochemical studies of chiral H-1 antagonists of histamine: the resolution, chiral analysis, and biological evaluation of four antipodal pairs. The resolution of the H-1 antihistamines chloropheniramine, dimethindene, carbinoxamine, and mebrophenhydramine is described. The optical purity of antipodal products is investigated by chiral HPLC (use of alpha 1-acid glycoprotein and beta-cyclodextrin columns) and NMR (spectra of beta-cyclodextrin inclusion complexes). Configurational relationships among the group are reviewed and assignments are confirmed and extended by circular dichroism evidence. Affinity constants of antipodal pairs for guinea pig ileum and cerebellum sites, determined by gut bath and binding experiments respectively, are reported together with some in vivo tests in man for central effects. Results are discussed in terms of configurational requirements for activity and variations in antipodal potency ratios within the group.


Single and repeated doses of the vasodilator/beta-adrenergic antagonist, carvedilol, block cirazoline- and isoproterenol-mediated hemodynamic responses in the conscious rat. The purpose of this study was to evaluate the effects of carvedilol, a beta 1&2-adrenergic blocker and vasodilator, on cirazoline-mediated changes in arterial blood pressure and isoproterenol-mediated changes in heart rate after acute and chronic administration. Conscious, chronically instrumented male Sprague-Dawley rats were injected with carvedilol (1 mg/kg, IV), prazosin (0.3 mg/kg, IV), or propranolol (1 mg/kg, twice daily for 8 days. After administration of the first dose of carvedilol on day 1, the vasopressor response to cirazoline (60 +/- 3 mmHg predrug) and the isoproterenol-induced tachycardia (152 +/- 13 beats/min predrug) were blocked (e.g., 7 +/- 4 mmHg postdrug and 11 +/- 3 beats/min postdrug, respectively). After the administration of carvedilol on day 8, the cirazoline vasopressor response was 2 +/- 1 mmHg and the isoproterenol-induced tachycardia was 4 +/- 3 beats/min, indicating effective alpha 1- and beta-adrenergic blockade after chronic dosing with carvedilol. Prazosin blocked the cirazoline-induced vasopressor response on both days 1 and 8 but had no effect on the isoproterenol-induced tachycardia. Propranolol blocked the isoproterenol-induced tachycardia on both days 1 and 8 but had no effect on the cirazoline vasopressor response. These data indicate that only carvedilol effectively blocked both alpha- and beta-adrenergic hemodynamic responses and that the antagonism of these responses with carvedilol was not diminished after chronic dosing of twice-a-day treatment for 8 days.


Combined invasive and noninvasive study of left ventricular systolic and diastolic function following acute administration of cicloprolol to subjects with normal cardiac function. Cicloprolol is a new beta-blocking agent with high selectivity for beta 1 receptors and high intrinsic sympathomimetic activity. We studied the acute hemodynamic effects of cicloprolol in nine subjects with no evidence of left ventricular dysfunction who underwent cardiac catheterization for the evaluation of chest pain. All patients had normal coronary angiography and left ventriculography. Left ventricular pressure was determined throughout the cardiac cycle using a Millar 8Fr Minotip catheter; an echocardiogram, phonocardiogram, and ECG were simultaneously recorded to obtain left ventricular pressure-diameter loops. All the measurements were repeated before and after the intravenous administration of cicloprolol. Cicloprolol was administered at increasing doses of 0.05, 0.10, and 0.25 mg/kg until a cardiac output increase of at least 15% over basal values was achieved. A decrease of mean arterial pressure or cardiac output after cicloprolol was not observed in any patient. Cicloprolol administration significantly increased cardiac output (24%), stroke volume (22%), and peak positive dP/dt (25%); no significant changes in heart rate, systemic blood pressure, right atrial pressure, or pulmonary artery pressures were observed. No significant change in the echocardiographic parameters occurred. Among the indices of left ventricular diastolic function, the time constant of isovolumetric relaxation was significantly decreased (-43%) after cicloprolol; moreover, the left ventricular pressure-diameter loop in the protodiastolic phase was shifted to the left following cicloprolol infusion. This study confirms that in subjects with normal left ventricular function cicloprolol can improve resting left ventricular systolic function, and it shows that this action can also be attended by a more rapid isovolumetric relaxation, similar to what has been observed with other sympathomimetic amines.


Dose-dependent effects of 6-hydroxy dopamine on deprivation myopia, electroretinograms, and dopaminergic amacrine cells in chickens. We found that a single intravitreal injection of 6-hydroxy dopamine (6-OHDA) is highly efficient in blocking the development of deprivation-induced myopia in young chickens. To investigate the effects of 6-OHDA on retinal function, we studied electroretinograms (ERGs) in chickens aged 15-25 days, 4 days subsequent to the injection. Both spectral sensitivity and oscillatory potentials were tested. In addition, a histological examination was performed of dopaminergic amacrine cells labeled by a monoclonal antibody against tyrosine hydroxylase. We found that, at doses of 6-OHDA sufficient to suppress deprivation myopia entirely, no effect could be detected on either the ERGs or on the density and appearance of dopaminergic amacrine cells. For higher doses, spectral sensitivity and the number of dopaminergic amacrine cells declined gradually. In contrast, as doses increased, oscillatory potentials 1 and 2 grew in amplitude only to decline at the highest doses. The results indicate that (1) development of deprivation myopia requires normal retinal function and that (2) slight changes in the gains of dopaminergic pathways are sufficient to block the development of deprivation myopia.


Triphasic sequence of neointimal formation in the cuffed carotid artery of the rabbit. A nonocclusive silicone cuff placed around the rabbit carotid artery results in a diffuse intimal thickening. The early stages of this phenomenon were studied by light microscopy, immunohistochemistry, and electron microscopy. Neointimal formation appeared to be triphasic. The first phase started 2 hours after cuff placement, with vascular infiltration by polymorphonuclear leukocytes (PMNs). In the second phase, starting within 12 hours, 1.90 +/- 0.36% of the medial smooth muscle cells (SMCs) were replicating, as demonstrated by their immunoreactivity for proliferating cell nuclear antigen (PCNA). The third phase was characterized by the appearance, from day 3 onward, of subendothelial SMCs that were immunoreactive for alpha-SMC actin and vimentin. A few cells showed immunoreactivity for PCNA. During this phase all the PMNs disappeared, but SMC replication in the media was still present, as indicated by the presence of mitoses and the persisting immunoreactivity for PCNA (0.76 +/- 0.22% at day 7). In the third phase the number of subendothelial cells increased (104 +/- 15 SMC nuclei per section at day 7, of which 8.89 +/- 2.26% were PCNA-positive) and was associated with deposition of collagen type IV and fibronectin. At 14 days a complete, circular neointima was present and contained 2.13 +/- 0.28% replicating SMCs. The media showed 0.44 +/- 0.08% cell-cycling SMCs, which was still four times higher than normal. During the first week there was also a significantly higher PCNA activity in the media of sham-operated carotid arteries (no cuff present) than in nonsurgical ones. However, this did not lead to the formation of a neointima. We conclude that in the cuff system SMC replication in the media precedes the neointimal formation. The system can be used to study SMC replication, migration, and neointimal formation with minimal medial SMC damage.


Predictors of the course of tardive dyskinesia in patients receiving neuroleptics. As many patients still require maintenance neuroleptic medication, it is important to determine what factors affect the course of tardive dyskinesia (TD) during ongoing neuroleptic treatment. In this study the data are from a series of annual examinations using the Abnormal Involuntary Movement Scale (AIMS) in a cohort of outpatients. Average neuroleptic exposure at the commencement of the examinations was 10 yr. The data on AIMS scores at successive visits are analysed using autoregressive modeling. Two hundred thirty-five patients contributed the 678 pairs of examinations. Separate analyses are reported for orofacial, limb-truncal, and total AIMS scores. Different predictor variables emerged as important for orofacial and limb-truncal dyskinesia. Age, sex, being on anticholinergic medication, and two variables describing duration of neuroleptic exposure contributed to the outcome score for total AIMS score after 1 yr.


Loss of the Kamin blocking effect in acute but not chronic schizophrenics. Differences between research diagnostic criteria (RDC)-diagnosed acute and chronic schizophrenics and normal controls were studied using a Kamin blocking procedure. Blocking is an established animal learning procedure, thought by some researchers to reflect selective attention; decreased blocking indicates increased processing of irrelevant stimuli. It was predicted that this pattern would be obtained in acute schizophrenics, tested soon after admission, for two reasons: (1) evidence from previous clinical studies indicates that acute schizophrenics are more aware of nonsalient aspects of their environment than controls; and (2) blocking is disrupted in animals in a hyperdopaminergic state and restored by neuroleptic medication. This was the case: acute, but not chronic, schizophrenics showed disrupted blocking. This disruption was especially clear in those acute schizophrenics tested within 2 weeks of hospital admission. By the second test session (in a cross-over design), there was some evidence of normalization in performance in the acute schizophrenics. These findings are considered with regard to the dopamine hypothesis of schizophrenia.


Drug distribution between blood and brain as a determinant of antipsychotic drug effects. Concentrations of the neuroleptics haloperidol, bromperidol, fluphenazine, chlorpromazine and its metabolites nor-1- and nor-2-chlorpromazine, thioridazine and its metabolites mesoridazine, sulforidazine, and northioridazine, and promazine were estimated in serum and brain of rats by high performance liquid chromatography (HPLC) with electrochemical detection following 5 days of chronic administration of drug at typical doses (haloperidol, bromperidol, and fluphenazine 1 mg/kg/day; chlorpromazine, promazine, and thioridazine 25 mg/kg/day). The observed ratio of brain-to-serum concentration of drug varied widely (0.18-62.5) among neuroleptics studied. High potency agents had more favorable brain-to-blood distribution than low potency agents, and a strong correlation (r = 0.734, p < 0.05) was observed between the brain-to-serum ratios of the neuroleptics and standard clinical doses of drug. This finding suggests that drug distribution is a significant determinant of clinical potency. For most neuroleptics, including drugs with high (fluphenazine, haloperidol) and low potency (thioridazine) such as dopamine D2 antagonists, concentration of drug in the brain was similar. If the results are applicable to patients, they suggest that the degree of dopamine D2 blockade achieved during treatment may vary by drug. Chlorpromazine and promazine were notable for producing high concentrations of drug in the brain at typical doses, suggesting that optimal doses might be lower than those in common use. These results may be important in designing and interpreting studies of the effects of neuroleptic drugs in animals and patients.


Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistant phenotype. The multidrug-resistance gene, MDR1 is expressed in many normal tissues, but little is known about its expression in normal hematopoietic cells. Using the monoclonal antibody C219 and flow cytometric analysis, P-glycoprotein (P-gp) was found to be expressed in all peripheral blood (PB) subpopulations (CD4, CD8, CD14, CD19, CD56) except granulocytes. To specifically determine MDR1 gene expression, these PB subpopulations were isolated by fluorescence-activated cell sorting (FACS) and analyzed for MDR1 mRNA by polymerase chain reaction (PCR). All subsets were positive by PCR, but only minimal MDR1 mRNA was detected in monocytes and granulocytes. Significant efflux of Rhodamine-123 (Rh-123), a measure of P-gp function, was detected in CD4+, CD8+, CD14+, CD19+, and CD56+ cells but not in granulocytes. Next, PCR-analysis was performed on FACS-sorted bone marrow (BM) cells to assess MDR1 expression in different maturational stages. Precursors (CD34+), early and late myeloid cells (CD33+/CD34+, CD33+/CD34-) as well as lymphocytes of the B-cell lineage (CD19+/CD10+, CD19+/CD10-) expressed the MDR1 gene. BM monocytic cells (CD33++/CD34-) were negative, and a very weak signal was detected in erythroid cells (glycophorin A+). Significant Rh-123 efflux was found in CD34+, CD10+, CD33+, and CD33++ BM cells, but not in glycophorin A+ cells. We conclude that PB and BM lymphocytes, PB monocytes, BM progenitors, and immature myeloid cells, but not late BM monocytes, erythroid cells, and PB granulocytes, express MDR1 mRNA and a functional P-gp. These results have to be taken into account when MDR1 expression is determined in tumor samples containing normal blood cells.


Expression and activity of the multidrug resistance P-glycoprotein in human peripheral blood lymphocytes. P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump for different lipophilic compounds, including many anticancer drugs and fluorescent dyes. We have previously reported that the efflux of fluorescent dyes from lymphoid cells of human bone marrow was directly correlated with the cellular P-gp content. In the present study, we show that human peripheral blood lymphocytes (PBL) also express P-gp, and that P-gp expression correlates with the efflux of fluorescent dyes from PBL. This efflux was suppressed not only by chemical inhibitors of P-gp but also by a P-gp-specific monoclonal antibody UIC2, thus providing direct evidence that it was mediated by P-gp. We have also characterized dye efflux and UIC2 reactivity in specific PBL subsets. P-gp was expressed in the majority of CD56+, CD8+, and CD20+ lymphocytes, but in less than one half of CD4+ cells. P-gp-mediated dye efflux was highly heterogeneous relative to the expression of CD56RA, CD56RO, Leu-8, and HLA-DR antigens. No significant P-gp activity was detectable in CD14+ monocytes. MDR1 expression in normal lymphocytes may be a determinant of multidrug resistance in the corresponding malignancies.


Alterations of bone marrow sinus endothelium induced by ionizing irradiation: implications in the homing of intravenously transplanted marrow cells. The endothelium of bone marrow sinuses is a continuous layer which is selective in its cellular transport. It is not known how selective and massive seeding of hemopoietic progenitor cells after intravenous transplantation of marrow cells occurs. We postulate that the conditioning irradiation could disrupt the endothelial barrier, thus permitting the "homing" of progenitor cells to occur. To demonstrate this phenomenon, we irradiated mice with doses ranging from 100-2000 cGy-total body and studied perfusion-fixed marrow by transmission electron microscopy. The major finding was sloughing and denudation of plasma membrane, particularly on the luminal side of endothelium. Membrane vesiculation was also frequently seen in this border. Moreover, dilatation of the perinuclear space and rough endoplasmic reticulum was commonplace and testified to instability and fragility of the membrane system. Focal cytoplasmic swelling of endothelium was seen reflecting increased permissiveness of the endothelial barrier. Endocytosis and phagocytosis were increased in the marrow; and the endothelium, normally quiescent with regard to phagocytosis, was now overtly phagocytic. A dipogenecity of the adventitial layer was increased as hemopoietic function of marrow decreased. The end result of membrane alterations in the endothelium was the appearance of discontinuities in these cells, which form the essential element of bone marrow-blood barrier. Consequent to these discontinuities, the permissiveness of the endothelial barrier was enhanced and those cellular elements, such as mature, nonreticulated erythrocytes that are normally confined to the vascular space, now appeared in large number in the hemopoietic compartment. With low doses, these findings were transient and repair set in by 1-2 weeks. With higher doses, total disruption of marrow-blood barrier occurred and the process did not seem to be repairable. We conclude that the conditioning irradiation before bone marrow transplantation is essential in disrupting the endothelial barrier and permitting large-scale entry of transplanted cells into the hemopoietic compartment.


The human hematopoietic stem cell in vitro and in vivo. A quantitative assay for a primitive human hematopoietic cell has been developed. The cell identified has been assigned the operational designation of long-term culture (LTC)-initiating cell based on its ability when cultured on supportive fibroblast monolayers to give rise to daughter cell(s) detectable by standard in vitro colony assays. Three lines of evidence support the view that the LTC-initiating cell assay may allow the relatively specific enumeration of totipotent cells with in vivo reconstituting potential. These involve the demonstration: (1) that conditions in analogous murine long-term cultures stimulate the extensive amplification (self-renewal) of some totipotent long-term repopulating cells, (2) that most of the LTC-initiating cells in normal human bone marrow are phenotypically different from most of the colony-forming cells present in the same cell suspensions in their possession of a number of characteristics specifically associated with transplantable stem cells; and (3) that cultured marrow cells from patients with chronic myeloid leukemia which, after maintenance under LTC conditions for 10 days contain some normal LTC-initiating cells but no detectable leukemic LTC-initiating cells, can after autografting reconstitute the hematopoietic system with normal cells.


Selective uptake of the somatostatin analog RC-160 across the blood-brain tumor barrier of mice with KHT sarcomas. The development of somatostatin analogs with anti-tumor effects has raised hopes for their use in various cancers and tumors of the central nervous system. However, for many therapeutic agents, access to normal brain is retarded by the blood-brain barrier (BBB) and to tumor tissues by a blood-brain tumor barrier (BBTB). We examined the ability of RC-160, a somatostatin analog with known anti-tumor activity, to cross the normal BBB and the BBTB in mice with brain sarcomas. In comparison with the normal BBB, the BBTB was about 10 times more permeable to the vascular marker albumin (radioactively labeled with 99mTc), but the BBTB still represents a substantial barrier. By contrast, the entry rate of RC-160, radioactively labeled with 125I, into brain sarcomas was 60 times higher than into normal brain tissue; more than 1% of the RC-160 injected i.v. was taken up by each gram of brain tumor. These results show that a brain tumor can selectively accumulate the potentially therapeutic agent RC-160.


Comparative studies on the mode of action of proctolin and phorbol-12,13-dibutyrate in their ability to contract the locust mandibular closer muscle. The role of proctolin has been further investigated in the locust (Locusta migratoria) mandibular closer muscles. Radioactive calcium uptake measurements were made using protease-dissociated muscle cells. Both the phorbol ester, phorbol-12,13-dibutyrate, and proctolin produce tonic contractions which are associated with the influx of extracellular calcium. The thresholds for proctolin and the phorbol ester to contract the muscle were 1-10 nM and 10-100nM, respectively, while their respective thresholds for evoking measurable calcium influx into the muscle cells were 0.1-1 nM for proctolin, and 0.1-1 pM for phorbol-12,13-dibutyrate. The effect of phorbol-12,13-dibutyrate is blocked by a number of protein kinase inhibitors (at a concentration of 0.1 mM), suggesting that an activation of a protein kinase can lead to calcium influx. These inhibitors, however, do not block the effect of proctolin, indicating that these two compounds work through different pathways, possibly converging on the same final target. In light of this finding, a number of other compounds have been tested to try to ascertain how proctolin mediates an increased calcium influx.


Resistance mechanisms to topoisomerase poisons: the application of cell culture methods. A survey is presented of two types of resistance to drugs acting on cellular topoisomerase enzymes, that occurring by altered drug transport and that occurring by altered target interaction. Particular attention is paid to the use of pairs of diagnostic drugs, one susceptible and one refractory to a particular resistance mechanism, in the determination of such resistance in cultured cells. Two pairs of drugs, each including the antitumor agent amsacrine, are used in the analysis of a number of cell lines. Also it is suggested that some normal hemopoietic precursor cells may exhibit two types of multidrug resistance, and that this should be taken into account in determining selectivity of topoisomerase-directed drugs for tumor cells.


Activation of MDR1 (P-glycoprotein) gene expression in human cells by protein kinase C agonists. P-glycoprotein, encoded by the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump for various lipophilic compounds. MDR1 is expressed in several types of normal human tissues and in a variety of tumors, where its expression has been correlated with resistance to chemotherapy. Some P-glycoprotein-overexpressing multidrug-resistant cell lines contain elevated amounts of protein kinase C (PKC). PKC activation was shown to increase the level of drug resistance in several cell lines, but the functional association of PKC with P-glycoprotein-mediated multidrug resistance remains unclear. We have studied the effects of lymphocyte-activating agents on P-glycoprotein activity in normal human lymphocytes, and found that 12-O-tetradecanoylphorbol-13-acetate (TPA), an efficient agonist of PKC, increased the activity as well as the levels of P-glycoprotein in these cells. TPA also increased P-glycoprotein expression in several cell lines derived from different types of leukemias and solid tumors. The increase in MDR1 gene expression was observed at both the protein and RNA levels. Induction of MDR1 mRNA was apparent as early as two hours after the addition of TPA. Diacylglycerol (DAG), a physiological stimulant of PKC, also increased the expression of MDR1 mRNA and P-glycoprotein. The induction of MDR1 expression by TPA and DAG was suppressed by staurosporine, a protein kinase inhibitor. The results suggest that MDR1 gene expression in different cell types is regulated by a PKC-mediated pathway. This finding has implications for the emergence of multidrug resistance in vitro and in vivo.


A randomized trial of reduced doses of azidothymidine in Japanese patients with human immunodeficiency virus type 1 infection. Adverse reactions to the standard dose (1,200 mg/day-1,500 mg/day) of azidothymidine (AZT) are serious. An in vitro pharmacokinetic study of intracellular AZT-5'-triphosphate suggested the feasibility of a clinical trial with reduced doses of AZT. A randomized trial with reduced doses of AZT (group A; 400 mg/day, n = 15, group B; 800 mg/day, n = 13), was conducted enrolling 28 patients with human immunodeficiency virus infection. The effective rate of AZT on CD4+ lymphocyte counts was similar for both groups, but the duration of the effect of AZT was significantly longer in group A (p less than 0.05). In group B, adverse reactions were more frequently observed (p less than 0.01), and AZT was withdrawn or the dose was reduced more frequently (p less than 0.05). These results suggest that AZT at a dose of 400 mg/day is less toxic, and is more beneficial for long-term treatment.


Genetic mapping of the murine gene and 14 related sequences encoding chromosomal protein HMG-14. The high-mobility-group chromosomal protein HMG-14 preferentially binds to nucleosomal core particles of mammalian chromatin and may modulate the chromatin configuration of transcriptionally active genes. The human gene for HMG-14 has been localized to the Down syndrome region of Chromosome (Chr) 21 and may be involved in the etiology of this syndrome. Here we show, by means of genetic linkage analysis of interspecific and intersubspecific backcross mice, that the murine functional gene, Hmg14, is located on the distal end of mouse Chr 16, a region known to have conserved synteny with human Chr 21. In addition to the functional gene for HMG-14, both human and mouse genomes contain many related sequences that are probably processed pseudogenes. Here we map the locations of 14 Hmg14-related sequences in two mouse genomes. The 14 mapped loci are widely dispersed on ten chromosomes (Chrs 3, 5, 7, 9, 11, 12, 16, 17, 19, and X) and can be detected efficiently with a single cDNA probe. Thus, the Hmg14 multigene family is well suited to serve as genetic markers for other linkage studies in mice.


A detailed linkage map of subtelomeric murine chromosome 12 region including the situs inversus mutation locus IV. A mouse model is an invaluable tool to tackle genesis of human congenital diseases that have so far eluded human studies. Homozygote for the i.v. mutation, the murine Si/Col strain presents a left-right lateralization defect of thoracic and abdominal organs and heart defects very similar to human ones. This i.v. mutation has been mapped to the region between the Aat and Igh-C loci, suggesting the presence of an equivalent human gene in the human syntenic 14q3 region. A precise linkage map of the region is, therefore, of great interest since it will contribute to the genetic approach of the i.v. gene. Analysis of 242 back-cross progeny from Mus musculus (MAI) or spretus strains of mice and SI/Col mice has allowed mapping of the i.v. gene to a linkage group of eight markers. It includes four genes: Aat (alpha 1-antitrypsin), Ckb (creatine kinase, brain form), Crip (cysteine-rich intestinal protein), and Igh-C (immunoglobulin heavy chain constant region complex); three murine microsatellites: D12Mit6, D12Mit7, and D12Mit8; and one new marker, D12Mtp1, defined by a minisatellite human probe, pYNZ2. After analysis of the data by the LINKAGE program, the following multilocus map has been constructed: centromere-D12Mit6-6.9 cM-D12Mit7-1.7 cM-D12Mtp1-2.6 cM-Aat-5.0 cM-(Ckb, Igh-C)-0.4 cM-D12Mit8-0.4 cM-Crip-11.2 cM-i.v.-telomere. This map differs from the previous map in placing i.v. locus telomeric to Igh-C. D12Mit6 and D12Mit7 are now precisely mapped centromeric to the locus Aat. In addition, a new locus D12Mtp1 is located between Aat and D12Mit7.


Free radicals and neurotransmitters in gerbil brain. Influence of age and ischemia reperfusion insult. In gerbil brain, levels of hydroxyl radicals (OH.) and neurotransmitters such as glutamate, aspartate, GABA (gamma aminobutyric acid) are low at birth, reach a plateau and decrease with age. On the other hand, when gerbils are exposed to an ischemia reperfusion insult (IRI) the older animals have a higher stroke index and hydroxyl radical as well as glutamate and other neuromediators are concomitantly increased. This discrepancy is probably due to differences in the ability of old individuals to respond to oxidative stress. The still incompletely understood relationship between oxidative damage to proteins and accumulation of amino acids, which have an important role as neurotransmitters at physiologic concentrations, but may become neurotoxic at high concentrations, is discussed.


Free radicals, lipid peroxidation, SOD activity, neurotransmitters and choline acetyltransferase activity in the aged rat brain. The mechanism of aging is suggested to be related to oxygen free radicals. Free radicals, lipid peroxidation and SOD activity have been reported to be increased in the aged brain. A Japanese herbal medicine, Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960), which has scavenging activities against hydroxyl radicals, superoxide, 1,1-diphenyl-2-picrylhydrazyl radicals, carbon-centered radicals and alpha-tocopheroxyl radicals, decreased carbon-centered radicals and thiobarbituric acid reactive substances (TBARS) levels in the aged rat brain after a 3-week oral administration of 5% TJ-960 solution. TJ-960 elevated superoxide dismutase (SOD) activity in the cytosol fraction of the hippocampus and hypothalamus of aged rats. It decreased norepinephrine and 5-hydroxytryptamine (5-HT) levels in the hypothalamus and increased the 5-HT level in the cerebellum. TJ-960 treatment increased choline acetyltransferase activity in aged rats. As herbal medicines do not generally have harmful side effects, antioxidant TJ-960 appears to be a suitable prophylactic agent against some neuronal symptoms of aging.


Protein modification in aging.

Protein modification in aging. During aging a number of enzymes accumulate as catalytically inactive or less active forms. The age-related changes in catalytic activity are due in part to reactions of the protein with "active" oxygen species such as ozone, singlet oxygen, or with oxygen free radicals as are produced during exposure to ionizing radiation or to metal ion catalyzed oxidation (MCO) systems. The levels of oxidized proteins in cultured human fibroblasts from individuals of various ages and in liver and brain extracts of rats of different ages increase progressively with age, and in old rats can represent 30-50% of the total cellular protein. The age-related increase in oxidized protein in rat liver and brain tissue is accompanied by a loss of glutamine synthetase (GS) and glucose-6-P dehydrogenase (G-6-PDH) activities, and to a decrease in the level of cytosolic neutral protease activity which is responsible for the degradation of oxidized (denatured) protein. Of particular significance are the results of experiments showing that similar age-related changes occur in the gerbil brain and that these changes are accompanied by a loss of short-term memory as measured by the radial arm maze technique. Chronic treatment (intraperitoneal injections) of old animals with the free radical spin-trap reagent, N-tert-butyl-alpha-phenylnitrone (PBN) resulted in normalization of the several biochemical parameters to those characteristic of the young animals; coincidentally, the short-term memory index was restored to the young animal values. These results provide the first evidence that there is likely a linkage between the age-dependent accumulation of oxidized enzymes and the loss of physiological function.


Glutathione homeostasis in rats chronically treated with ethanol. Evidence for an increased hepatic GSH export in vivo. The influence of chronic ethanol feeding to rats on the hepatic glutathione (GSH and GSSG) system (synthesis, catabolism, export) and on the GSH and GSSG concentrations in extrahepatic tissues was investigated. Histological examination of livers from ethanol pretreated rats revealed a minor dilatation of the hepatic sinusoids. After ethanol administration the distribution pattern of gamma-glutamyltranspeptidase (enzymehistochemistry) was nearly unchanged, but the hepatic activity of this enzyme was increased. The ethanol pretreatment led to a decrease in hepatic GSH content. The hepatic activity of the GSSG-reductase were increased after ethanol treatment whereas the activities of the GSH synthesizing enzymes (gamma-glutamyl-cysteinyl-synthetase and GSH-synthetase) were not affected. A strong increase in sinusoidal GSH export was found in the ethanol-pretreated rats. The GSH- and GSSG concentrations of brain, lung, kidney and skeletal muscle were unchanged. It can be concluded that the ethanol-induced alteration of the hepatic GSH metabolism is caused mainly by changes of the sinusoidal membrane of the hepatocytes (direct effect of ethanol on the sinusoidal GSH carrier) leading to an increased GSH export into plasma. This effect should not due to an increased extrahepatic requirement for GSH.


The role of the promoter in the expression of the PCNA gene. G1-specific temperature-sensitive (ts) mutants of the cell cycle arrest in G1 after serum stimulation at the restrictive temperature. Under these conditions, the RNA levels of late growth-regulated genes (such as DNA polymerase alpha, PCNA, thymidine kinase, and core histones) are markedly decreased or even undetectable, while early growth-regulated genes (for instance, c-myc) are normally expressed, and certain promoters are actually super-induced. We have used the human PCNA gene transfected into TK-ts13 cells (a G1-specific ts mutant) to investigate whether the inhibition of gene expression caused by this type of growth inhibition occurs at a transcriptional or post-transcriptional level. Constructs were made in which the 5' and 3' flanking sequences of the human PCNA gene were replaced by the corresponding elements of the SV40 T antigen coding gene. Using these constructs and data from run-on assays and RT-PCR, we conclude that the failure of expression of the PCNA gene in G1-arrested TK-ts13 cells occurs at the transcriptional level.


Hepatitis C virus infection in patients with idiopathic hemochromatosis (IH) and porphyria cutanea tarda (PCT). The prevalence of HCV antibodies in IH and PCT patients was examined. It was found that both groups are characterized by increased incidence of HCV infection. These results suggest a possible connection between HCV and iron overload.


Effects of excess protein intake on nitrogen utilization in young men. The efficiency of nitrogen (N) utilization was studied in 12 young male subjects. Protein intake levels were adjusted from moderate (1.08 and 1.18 g protein/kg/day) to high (1.74 and 2.00 g protein/kg/day). All of the food was supplied in the form of a normal mixed Chinese diet. Six subjects were admitted to a metabolic unit at a time for 56 days, in two consecutive periods. The results indicate that a higher protein intake causes more N excretion in urine and feces. Biologic value (BV) and net protein utilization (NPU) were markedly decreased during the high protein intake (HPI) period. However, a significant increase in the N balance was found in the presence of excessive protein intake. Digestibility of protein seemed to increase during the HPI period, with the apparent digestibility of the dietary protein being about 83% to 90%. We conclude from this study that excessive N intake reduces the efficiency of N utilization, but still results in a positive N balance in adult human subjects.


Relationship between brain serotonin and calmodulin in young, genetically obese (ob/ob) mice. The possible relationships between altered brain serotonin and calmodulin contents on the development of obesity were studied. Eight groups of mice separated by differences in phenotype, sex and age were used in this study. The brain contents of tryptophan, serotonin, 5-hydroxyindoleacetic acid (5-HIAA) and calmodulin were assayed. The contents of brain tryptophan showed no significant differences in any of the mice. The amount of brain serotonin in obese mice was 82% higher than that in their lean counterparts at four weeks of age, but only 11% higher at eight weeks of age. Regardless of age and sex, brain serotonin was positively correlated to the brain calmodulin in the lean mice (r = 0.559, p < 0.01), yet this was not found in obese mice. There was a strong positive correlation between serotonin and 5-HIAA in all mice (r = 0.679, p < 0.001). The elevated amount of serotonin in the brain of four-week-old obese mice is suggested to have important effects on thermoregulation in young genetically obese mice. The results also suggest that abnormal brain serotonin synthesis in obese mouse regulated by calmodulin might interact with certain factors, such as calcium ions, to complete the activation of serotonin-synthesized enzymes in the development of obesity.


Inheritance of hypertrophic cardiomyopathy in Chinese--M-mode and two-dimensional echocardiographic analysis of 28 families. To determine the mode of inheritance and degree of penetrance of hypertrophic cardiomyopathy (HC) in Chinese, 132 family members of 28 probands with HC were assessed by M-mode and two-dimensional echocardiography. Of these 132, 103 cases were first-degree relatives of the probands. Twenty-seven (20.4%) family members, including 19 cases of first-degree relatives of the probands, had HC. Familial occurrence of HC was noted in 13 (46.4%) families. In 10 families, the affected relatives were identified in successive generations, and the mode of inheritance was most consistent with an autosomal dominant trait. The frequency with which HC was identified in relatives increased significantly with the number of subjects studied and a positive family history of sudden death. Subgroup analysis, using multivariate logistic regression analysis, revealed that increasing age was independently associated with a higher frequency of definite cases in first-degree relatives of the probands. In contrast, by multivariate analysis, there were no significant differences between frequency of definite cases in female and male relatives or in different familial relationships (parent, sibling, offspring) to the probands.


Comparison of sodium transport processes of human and rat erythrocytes in hypertension. Erythrocytes of normotensive and hypertensive humans, as well as Sprague-Dawley, Wistar-Kyoto and spontaneously hypertensive rats, were prepared to have similar ionic compositions adequate for determining the activities of lithium-sodium (Li-Na) countertransport and sodium (Na) pump. The rate of Li-Na countertransport was significantly higher in erythrocytes of hypertensive subjects. This activity was not detected in rat erythrocytes, at two different ages, and over a six-fold of lithium (Li) content. Activities of Na pump were not significantly different among various groups. Human cells, in general, had a higher activity than rat cells; among rat cells, spontaneously hypertensive rats had a higher rate of sodium pump. Both Na-independent Li efflux and ouabain-insensitive Na efflux were significantly higher in rat erythrocytes than in human cells. Thus, employing the same methods, we have determined that while the properties of Na pump were similar in human and rat erythrocytes, Li-Na countertransport did not operate in the erythrocytes of either normotensive or hypertensive rats.


Antithrombin III activity in normal and toxemic pregnancies. From August 1989 to October 1990, 83 pregnant Chinese women were the subjects for measuring the levels of plasma functional antithrombin III (AT III) activity. The correlations of AT III activity with perinatal outcome and the changes in maternal hepatorenal function were analyzed. The population was divided into four groups: Group I (n = 30), normal pregnancies; Group II (n = 23), mild pre-eclampsia; Group III (n = 26), severe pre-eclampsia; and Group IV (n = 4), eclampsia. The results demonstrated that: 1) AT III activity decreased with the severity of toxemia (p < 0.001), 2) AT III activity correlated with the degree of perinatal outcome and maternal morbidity, and 3) reduction of AT III activity correlated with impairment of maternal hepatorenal function. In conclusion, plasma AT III activity is a valuable parameter in the evaluation of toxemia.