Comparative analyses of multi-species sequences from targeted genomic regions.

The systematic comparison of genomic sequences from different organisms represents a central focus of contemporary genome analysis. Comparative analyses of vertebrate sequences can identify coding and conserved non-coding regions, including regulatory elements, and provide insight into the forces that have rendered modern-day genomes. As a complement to whole-genome sequencing efforts, we are sequencing and comparing targeted genomic regions in multiple, evolutionarily diverse vertebrates. Here we report the generation and analysis of over 12 megabases (Mb) of sequence from 12 species, all derived from the genomic region orthologous to a segment of about 1.8 Mb on human chromosome 7 containing ten genes, including the gene mutated in cystic fibrosis. These sequences show conservation reflecting both functional constraints and the neutral mutational events that shaped this genomic region. In particular, we identify substantial numbers of conserved non-coding segments beyond those previously identified experimentally, most of which are not detectable by pair-wise sequence comparisons alone. Analysis of transposable element insertions highlights the variation in genome dynamics among these species and confirms the placement of rodents as a sister group to the primates.

Characterization of the DIM series of BALB/c preneoplasms for mouse mammary tumor virus-mediated oncogenesis.

The DIM series of preneoplasms, developed from the BALB/c mammary cell line COMMA-D, and DIM-derived tumors were examined for evidence of mouse mammary tumor virus (MMTV) involvement in the tumorigenic process. DIM tissues were shown to be free of exogenous MMTV inasmuch as Southern blot analysis of DNAs extracted from DIM preneoplasms and tumors showed the lack of a PstI-generated 4.2-kilobase MMTV-gag-pol band. To examine the possibility of endogenous MMTV action via enhancer insertion, DNAs from DIM preneoplasms and tumors were restricted with EcoRI, BamHI, or SstI and subjected to Southern blot analysis using an MMTV-long terminal repeat probe. The normal endogenous proviral content was detected in all DIM tissues assayed; no new proviruses were found. To examine the possibility of endogenous MMTV gene product oncogenesis, MMTV transcripts were quantified by slot blot analysis and MMTV envelope proteins were assayed by immunohistochemical staining of DIM tissue sections. While all DIM tissues expressed MMTV-long terminal repeat transcripts, the level of expression did not correlate with tumorigenicity. Most frequently, MMTV-env-containing sequences were more abundant than the 1.6-kilobase long terminal repeat transcript, the latter being the most promising candidate for a MMTV gene product mediating mammary tumorigenesis. However, preneoplasms and tumors of only the DIM 4 line contained levels of MMTV Mr 52,000 or 36,000 glycoproteins detectable by immunoperoxidase staining. Electron microscopy did not reveal any virus particles in DIM 4 tissues. These data do not substantiate MMTV as a causative agent in the formation of the DIM preneoplasms or tumors. Thus while MMTV function may confuse the interpretation of causative events in the formation of other preneoplasms and neoplasms, the DIM preneoplasms represent a model system for the study of MMTV-independent mechanisms.

Overexpression of oncogene products can cause tumor progression without parenchymal infiltration in the rat brain.

Tumor cell progression and parenchymal infiltration play important roles in the pathogenesis of gliomas. Using retrovirally marked rat 9L gliosarcoma cells, which when injected in the CDF rat brain form noninfiltrating tumors which grow only surrounding existing blood vessels, we were able to investigate elements of tumor growth and progression within the substance of the brain. Transfection of 9L by oncogenes associated with tumor progression and expressed in gliomas resulted in alterations in the in vivo phenotype of these cells, with the production of faster growing, well-vascularized, large solid tumors. However, diffuse infiltration was not seen. Moreover, coinjection of 9L or its transfectants together with the infiltrative C6 cell line resulted in a mixed tumor of noninfiltrating 9L cells in an environment of infiltrating C6 cells, suggesting that the infiltrative ability of C6 cells is controlled on the individual cell level.

Some hypothalamic hamartomas contain transforming growth factor alpha, a puberty-inducing growth factor, but not luteinizing hormone-releasing hormone neurons.

Activation of LH-releasing hormone (LHRH) secretion, essential for the initiation of puberty, is brought about by the interaction of neurotransmitters and astroglia-derived substances. One of these substances, transforming growth factor alpha (TGFalpha), has been implicated as a facilitatory component of the glia-to-neuron signaling process controlling the onset of female puberty in rodents and nonhuman primates. Hypothalamic hamartomas (HH) are tumors frequently associated with precocious puberty in humans. The detection of LHRH-containing neurons in some hamartomas has led to the concept that hamartomas advance puberty because they contain an ectopic LHRH pulse generator. Examination of two HH associated with female sexual precocity revealed that neither tumor had LHRH neurons, but both contained astroglial cells expressing TGFalpha and its receptor. Thus, some HH may induce precocious puberty, not by secreting LHRH, but via the production of trophic factors--such as TGFalpha--able to activate the normal LHRH neuronal network in the patient's hypothalamus.

Myasthenic syndrome studied with single fiber electromyography.

A patient with the myasthenic syndrome was investigated with single fiber electromyography (SFEMG). It was found that 35 of 36 potential pairs had increased "jitter" and that 29 of these demonstrated impulse blockings. There was improvement in neuromuscular transmission with higher rates of innervation. Transmission worsened following rest. The SFEMG findings at the motor end-plate level correlate well with the results of repetitive stimulation and in vitro studies.

Myasthenia gravis with features of the myasthenic syndrome. An investigation with electrophysiologic methods including single-fiber electromyography.

A 47-year-old woman had myasthenia gravis with only moderate weakness and a consistently poor response to anticholinesterase medication. She was investigated with routine electrophysiologic studies and single-fiber electromyography. The studies showed a limited effect of anticholinesterase medication. There was a decrement to 2 Hz stimulation of not less than 25 percent regardless of medication and administration of edrophonium. With faster rates of stimulation (10 to 20 Hz), there was a facilitation up to 190 percent. In addition, with postcontractive or post-tetanic potentiation the action potential in the abductor digiti minimi muscle was increased from the initial value of 2.5 mv or less to up to 6 mv. Single-fiber electromyographic investigations showed increased jitter and blocking with little response to drugs, as well as facilitation in a large number of motor end-plates in association with both voluntary activity and repetitive stimulation. The findings indicated a mixture of phenomena seen in myasthenia gravis and the myasthenic syndrome.

Evidence of subnormal function of association cortex in presenile dementia.

Regional cerebral blood flow (rCBF) was measured in 23 patients with organic dementia (17 patients with Alzheimer's disease and six with low-pressure hydrocephalus [LPH]). The flow of gray matter was significantly reduced, especially in occipito-parieto-temporal regions, as well as frontally in some patients. During activation with psychologic tests, reading, etc., the flow augmentation in the association areas in the demented patients was not as marked as in nondemented controls. Instead, in some patients, a flow diminution was recorded in the same regions (intellectual steal). Organic dementia appears to be accompanied by a low activity in the association cortex and a reduced ability to activate these regions during mental effort.

Neostigmine-induced end-plate proliferation in the rat. A study using supra-vital methylene blue.

The supra-vital methylene blue technique was used to study motor end-plate morphology in 21 BD-1X rats, treated for 7 to 16 weeks with oral neostigmine bromide, and in five control animals. Motor endings of treated animals showed preterminal, terminal, and ultraterminal sprouting and an increase in mean end-plate length (p less than 0.001). These changes were most prominent after 14 weeks of treatment. After 16 weeks of therapy, mean end-plate length decreased and the proliferative abnormality became less evident. The adverse effect of neostigmine on motor end-plates may have clinical relevance in the management of myasthenia gravis and may be partly responsible for the end-plate abnormalities previously reported in this disease.

Interictal psychopathology in patients with ictal fear: a quantitative investigation.

We assessed the hypothesis that psychopathology is overrepresented in patients with temporal lobe epilepsy (TLE) who experience ictal fear. Eleven such patients were matched with two control groups: patients who had TLE without ictal fear and patients who had generalized epilepsy. On several MMPI scales, the TLE-ictal fear group scored significantly higher than the two control groups, which did not differ significantly from each other. Furthermore, the TLE-ictal fear group had more MMPI clinical scales that were pathologically elevated, had significantly more patients with histories of admission to psychiatric hospitals, and had more MMPI profiles that were psychotic than either of the control groups.

Clinical utility of liver biopsy in patients with serum antibodies to the human immunodeficiency virus.

PURPOSE: Patients with the acquired immunodeficiency syndrome (AIDS) frequently have liver dysfunction, which may be due to a number of causes. Determination of the patients who are likely to benefit from liver biopsy, an invasive procedure, is therefore important. In this study, the results of liver biopsy in patients with AIDS were compared to those in human immunodeficiency virus (HIV)-infected patients without AIDS. PATIENTS AND METHODS: Thirty-six consecutive patients with antibodies to HIV present in the serum underwent liver biopsy from 1984 through 1988 at the Bronx Municipal Hospital. Twenty (56%) of the patients had AIDS diagnosed prior to the liver biopsy. Indications for the liver biopsy were unexplained fever in 83%, and abnormal serum levels of biochemical parameters of liver function in 89%. RESULTS: Liver biopsy was diagnostic in 18 cases (50%), including findings of hepatic infection by mycobacteria in 15, cytomegalovirus in two, and schistosoma in one; these infections had been previously detected at an extrahepatic site in only two cases. Helpful clinical information in 10 others included findings of granulomas of undetermined etiology in four, cirrhosis in five, and chronic persistent hepatitis in one. Patients with a diagnostic biopsy, as compared to patients with a nondiagnostic biopsy, had a statistically significant increase in the frequency of having AIDS diagnosed before the biopsy, longer duration of AIDS (in patients with AIDS diagnosed before the biopsy), greater number of different prior opportunistic infections, and a more elevated serum alkaline phosphatase level. For example, 70% of patients with AIDS, as compared to 25% of patients with serum antibodies to HIV but without AIDS, had diagnostic liver biopsies. Patients with a diagnostic biopsy also had statistically significantly more frequent pulmonary symptoms, possibly due to more frequent occurrence of Pneumocystis carinii pneumonia. In particular, the 15 patients with hepatic mycobacterial infection, as compared to the other patients, had a statistically significant increase in the frequency of having AIDS diagnosed prior to the biopsy, longer duration of AIDS, more frequent prior opportunistic infections, more severe leukopenia, and a more elevated serum alkaline phosphatase level. Liver biopsy was more sensitive than bone marrow aspiration and biopsy at detecting mycobacterial infection. CONCLUSION: Liver biopsy, when indicated, is useful to detect opportunistic infection in HIV-infected patients who are not known to have AIDS.

The wasted leg syndrome, a single fibre electromyographic study of arms and legs.

Single fibre electromyography (SFEMG) was performed on 11 patients with the wasted leg syndrome. Five were of South Asian origin and the others Chinese. In most cases the disorder progressed for about 5 yr before becoming static or very slowly progressive. The SFEMG fibre density was increased similarly in both the affected and less affected leg, but was normal in the arms except for a few longstanding cases. The limited extent of clinical and electrophysiological abnormalities suggests that the wasted leg syndrome is a distinct entity.

Why is the gracilis muscle relatively uninvolved in neuromuscular disorders?

CT scanning of thigh muscles has shown that the gracilis is often relatively resistant to degeneration in neuromuscular disorders. We have compared the histometric features of the gracilis muscle with the biceps femoris and vastus lateralis muscles in the right and left thighs of 12 subjects without neuromuscular disease. There was striking variation between individuals, but marked similarities between the two limbs in individuals. The gracilis and vastus lateralis muscles contained fewer Type 1 fibres than the biceps femoris. The Type 2 fibres were slightly larger in vastus lateralis than in the other two muscles. There is thus no evident morphological difference between the gracilis muscle and other thigh muscles that can be correlated with the different susceptibility of these muscles in neuromuscular diseases. However, these muscles are subject to differing physiological stresses in movement, and these may be important in determining the response to disease.

Adrenergic agents. 3. Synthesis and adrenergic activity of some catecholamine analogs bearing a substituted sulfonyl or sulfonylalkyl group in the meta position.

The m-phenolic group of catecholamine beta-adrenergic agonists may be replaced by various functionalities capable of undergoing H bonding. Considerable latitude in the nature of the OH simulating group is permissible with retention of activity; however, the most extensively studied analogs are ones in which a mobile proton is attached to an O or N atom. In a search for new selective bronchodilators a series of catecholamine analogs bearing a substituted sulfonyl or sulfonylalkyl group in the meta position (i.e., groups in which the mobile H is attached to a C atom) was examined. These compounds were studied for beta-adrenergic agonist activity in vitro by measuring their ability to relax tracheal smooth muscle and to increase the rate of spontaneously beating right atria of guinea pigs. Adrenergic activity was influenced by the nature of the alkylene bridge between the sulfonyl and aromatic groups, branching of the ethanolamine side chain, stereochemistry, and substitution of the sulfonyl and amino groups. Beta-adrenergic blockage was noted for some compounds having the sulfonyl attached directly to the ring. Greatest beta-adrenergic agonist potency and tissue selectivity was observed with a m-MeSO2CH2 substituent. One of these compounds, alpha-[[(1,1-dimethylethyl)amino]methyl]-4-hydroxy-3-[(methylsulfonyl)methyl]benzenemethanol hydrochloride (sulfonterol hydrochloride, USAN), was studied more extensively in animals and is presently being examined for bronchodilator activity in man.

Adrenerigic agents. 7.1 Synthesis and beta-adrenergic agonist activity of several 2-pyridylethanolamines.

In a search for new selective bronchodilators, three 2-pyridylethanolamines, i.e., 2-tert-butylamino-1-(5-hydroxy-2-pyridyl)ethanol (2b), a related 6-methylsulfonylmethyl (2c), and, a 6-methyl (2d) derivative, were prepared. These compounds were examined for potential bronchodilator activity in an in vitro test for relaxation of guinea pig tracheal tissue. Potential cardiac stimulant activity was evaluated in vitro by measuring changes in the rate of spontaneously beating guinea pig right atrial muscle. Comparison of potency in the tracheal test relative to that in the atrial procedure provides a measure of selectivity. Results of this study indicate that replacement of the phenyl ring of a para-hydroxylated phenylethanolamine with a 2-pyridyl system generally results in compounds which retain a high order of potency in the tracheal test; however, selectivity for tracheobronchial vs. cardiac tissue is markedly greater for the pyridyl derivatives. The alpha-picoline, 2-tert-butylamino-1-(5-hydroxy-6-methyl-2-pyridyl) ethanol (2d), which bears labile protons at a position meta to the ethanolamine side chain, was about equipotent with the corresponding 6-unsubstituted relative 2b. The reason for the failure of these apparently appropriately located labile protons to enhance beta-adrenoreceptor agonist activity is uncertain.

Adrenergic agents. 8.1 Synthesis and beta-adrenergic agonist activity of some 3-tert-butylamino-2-(substituted phenyl)-1-propanols.

Replacement of the benzylic hydroxyl group of N-tert-butylnorepinephrine with a hydroxymethyl substituent affords a propanolamine homologue which retains a high degree of beta-adrenergic agonist activity. As modification of the meta substituent of catecholic ethanolamines, such as N-tert-butylnorepinephrine, often provides compounds that exert a more pronounced effect in relaxing tracheobronchial smooth muscle (beta2-adrenergic agonist) than in stimulating cardiac muscle (beta1-adrenergic response), a series of 3-tert-butylamino-2-(3-substituted 4-hydroxyphenyl)-1-propanols was prepared. The 3-meta substituents included HOCH2 (1b), H2NCONH (1c), MeSO2NH (1d), H (le), and NH2 (1f). These phenylpropanolamine derivatives were compared with their phenylethanolamine counterparts in in vitro tests that measure the ability of these compounds to relax spontaneously contracted guinea pig tracheal smooth muscle (a measure of potential bronchodilating activity) and to increase the rate of contraction of a spontaneously beating guinea pig right atrial preparation (an indicator of potential cardiac stimulating activity). In these tests all of the propanolamine derivatives included in the study were less potent than their ethanolamine relatives. In both series replacement of the catecholic m-hydroxyl group with the indicated substituents usually resulted in compounds with increased selectivity for tracheobronchial vs. cardiac muscle.

Syntheses and in vitro evaluation of 4-(2-aminoethyl)-2(3H)-indolones and related compounds as peripheral prejunctional dopamine receptor agonists.

A series of (beta-aminoethyl)indolones and related compounds was synthesized and evaluated in vitro as peripheral prejunctional dopaminergic agonists in the field-stimulated isolated perfused rabbit ear artery. 4-[2-(Di-n-propylamino)ethyl]-7-hydroxy-2(3H)-indolone was the most potent compound (ED50 = 2 +/- 0.3 nM) tested, while the related secondary amine 24 and the des-OH derivatives 28 and 34 were only slightly less potent. 4-Methoxybenzeneethanamine and 2-methyl-3-nitrophenylacetic acid were employed as starting materials for for the synthesis of the 4-(beta-aminoethyl)indolones. The ring-opened 3-acylamino analogues 46 and 47 were prepared via nitration of the phenethylamine 43 derived from 4-methoxyphenylacetic acid. The inactive isomeric indolones 38, 39, and 41 were derived from 4-nitrobenzeneethanamine and from indolone-6-acetic acid.

Inhibitors of phenylethanolamine N-methyltransferase and epinephrine biosynthesis. 1. Chloro-substituted 1,2,3,4-tetrahydroisoquinolines.

In a search for inhibitors of epinephrine biosynthesis as potential therapeutic agents, a series of 13 ring-chlorinated 1,2,3,4-tetrahydroisoquinolines was prepared. These compounds were tested initially for their ability to inhibit rabbit adrenal phenylethanolamine N-methyltransferase (PNMT) in vitro. Enzyme-inhibitor dissociation constants, determined for the six most potent members of the series, indicated the following order of decreasing potency: 7,8-Cl2 greater than 6,7,8-Cl3 greater than 7-Cl approximately 5,6,7,8-Cl4 greater than 5,7,8-Cl3. These compounds were subsequently examined for PNMT-inhibiting activity in intact rats and mice. 7,8-Dichloro-1,2,3,4-tetrahydroisoquinoline (13, SK&F 64139) was the most potent member of the series both in vitro and in vivo and is currently undergoing clinical investigation.

Comparative pharmacokinetics of lovastatin, simvastatin and pravastatin in humans.

Twelve healthy male volunteers received single market-image 40-mg oral doses of lovastatin and simvastatin (both lactone prodrugs), or pravastatin (a beta-hydroxyacid) at 1 week intervals in a three-way crossover study to quantify HMG-CoA reductase inhibitors in plasma. Multiple plasma samples were collected up to 24 hours after the dose and assayed for active and total HMG-CoA reductase inhibitors. After equal oral doses, higher plasma concentrations of HMG-CoA reductase inhibitory activity after pravastatin than after either lovastatin of simvastatin (2-3 fold greater area under the concentration-time curve) suggest a greater potential availability of pravastatin-related inhibitory activity to peripheral tissues.

Lambert Eaton syndrome: autonomic neuropathy and inappropriate antidiuretic hormone secretion in a patient with small cell carcinoma of the lung.

A patient with small cell carcinoma of the lung developed asymptomatic autonomic neuropathy, inappropriate antidiuretic hormone (ADH) secretion and Lambert Eaton myasthenic syndrome. The autonomic neuropathy and inappropriate ADH secretion were present at the time of diagnosis of the tumour. Following chemotherapy these resolved, but 5 months later the patient developed Lambert Eaton syndrome which responded to 3,4-diaminopyridine. This sequence of changes appears to be unique.

Juvenile-onset bulbospinal muscular atrophy with deafness: Vialetta-van Laere syndrome or Madras-type motor neuron disease?

A girl with rapid-onset, bulbospinal muscular atrophy and deafness is described. The patient's mother and brother showed EMG features consistent with subclinical involvement. T is bulbospinal form of spinal muscular atrophy associated with deafness described by Vialetto and van Laere closely resembles the Madras type of motor neuron disease, also associated with deafness, described by Jagganathan and colleagues.