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PubMed1999

Applications of the yeast two-hybrid system.

In recent years, the yeast two-hybrid system has become the method of choice for detection and analysis of protein-protein interactions in an in vivo context. This system, which capitalizes on the significant genetic history and ease of protocols for manipulation of Saccharomyces cerevisiae, is accessible to most laboratories and is applicable to the pursuit of a large variety of experimental goals. To date, the two-hybrid system has seen widespread application for identification of interaction partners by screening methods using a particular protein of interest as a "bait." Large-scale ventures are also in progress, for example, a cataloging of interactions among the cellular proteins in yeast. However, this method also has tremendous potential for more focused analyses of specific proteins and should become more routine as an alternative or adjunct approach for many structure-function investigations.

Cloning, Molecular↗
PubMed2001

Mass spectrometry for the study of protein-protein interactions.

The identification of subpicomolar amounts of protein by mass spectrometry (MS) coupled with two-dimensional methods to separate complex protein mixtures is fueling the field of proteomics, and making feasible the notion of cataloging and comparing all of the expressed proteins in a biological sample. Functional proteomics is a complementary effort aimed at the characterization of functional features of proteins, such as their interactions with other proteins. Proteins comprise modular domains, many of which are noncatalytic modules that direct protein-protein interactions. Capturing proteins of interest and their interacting proteins by using high-affinity antibodies presents a simple method to prepare relatively simple protein mixtures easily resolved in one-dimensional formats. Individual or mixtures of proteins identified as stained bands in polyacrylamide gels are subjected to in situ digestion with the protease trypsin, and the extracted peptide fragments are analyzed by MS. The quality, quantity, and complexity of the tryptic digest, the species origin of the proteins, and the quality of the corresponding databases of genomic and protein information greatly influence the subsequent MS analysis in terms of degree of difficulty and methodological approach required to make an unambiguous protein identification. In this article we report the isolation of associated proteins from a complex cell-derived lysate by using an epitope-directed antibody. The protein pICLn engineered to carry an epitope tag was purified from cultured human embryonic kidney cells, and found to associate with a variety of proteins including the spliceosomal proteins smE and smG. By application of this general approach, the systematic identification of protein complexes and assignment of protein function are possible.

Algorithms↗
PubMed1996

Heterogeneity of papillomaviruses.

The classification of the papillomaviral sequence into types is satisfactory for many purposes, but the resulting catalog does not accurately depict the extent of diversification of this family of viruses. Phylogenetic and mutational analyses reveal that some viral forms are much more ancient than other forms. Episodic evolutionary events and zoonotic transmission may have contributed to the overall pattern. Convergent evolution appears to be the best explanation for the scattered occurrence across the family tree of viruses with high oncogenic risk. We look for the determinants of this risk among the less conserved residues of the viral E6 and E7 transforming proteins.

Amino Acid Sequence↗
PubMed1996

Patterns of spread of recurrent intraabdominal sarcoma.

A prominent site for recurrence of retroperitoneal and visceral sarcoma is the abdominal cavity. In an attempt to understand the causation of local and regional recurrence, 21 sarcoma patients who had previously undergone "complete" surgical removal of the primary tumor were prospectively studied. Data were obtained retrospectively from the first operation and prospectively from the reoperative procedure at the Washington Cancer Institute. At the primary and reoperative surgeries, 9 abdominopelvic regions and 21 sites were scored and then cataloged in a standardized fashion. Tumor locations and surgical resections were statistically analyzed in an attempt to establish patterns of recurrence within the abdomen and pelvis. There was a significant difference in sites of recurrence when sarcomas that involved the parietal structures were compared with those that involved small bowel. Peritoneal implants (nodular recurrences) were uniformly present in both groups. In contrast, resection site recurrences were very common with primary sarcomas invested by parietal peritoneum, while they were absent in those covered by visceral peritoneum. When primary surgeries were compared with reoperations, there was an increasing intraabdominal dissemination; the mean number of regions increased from 1.81 to 5.13. The change in distribution of sarcoma deposits at reoperation was greatest in right upper (because of liver surface) central and pelvic abdominopelvic regions and lowest in the left upper and epigastrium. The four anatomic sites that revealed a significant increase in involvement at the time of recurrence were the greater omentum, liver surface, large bowel, and the cul-de-sac of Douglas (all p < 0.002). Regions with tumor involvement or regions subjected to surgical trauma at the time of primary sarcoma resection were significantly more likely to show sarcoma deposits than to be sarcoma free at reoperation. These data taken together may suggest that sarcoma tumor emboli are frequently present in the abdomen at the time of resection of the primary cancer and that these tumor emboli are entrapped in fibrinous material at or immediately adjacent to sites of surgical trauma and along narrow margins of resection. Tumor cell entrapment of sarcoma emboli released into the peritoneal cavity prior to or at the time of sarcoma resection may help explain the distribution of nodular and fusiform recurrence of abdominopelvic sarcoma.

Abdominal Neoplasms↗
PubMed2002

Drugs, the human genome, and individual-based medicine.

The so-called "Genomic Revolution" has made possible the high-resolution sequencing of the DNA making up the human genome. One of the main conclusions of the currently available sequencing data is that individuals differ genetically from one another via sequence variations in their genomes. When affected genes are transcribed and translated, some of these sequence variations result in protein products that may affect the functioning of the proteins. This has led to widespread optimism that information on an individual's pattern of sequence variations will lead to drugs that target that individual's variant proteins and make "individual-based medicine" possible. I this chapter some of the assumptions underlying the proposed production of individual drug treatments are examined. The assumptions are viewed in the light of very recent experimental evidence about the sequence patterns found in humans. Also discussed are ancillary ethical problems in cataloging and using databases containing individuals' sequence data, what human genomic sequences are revealing about the use of animal models in developing drugs, and how evidence is mounting that the human genome is only one element serving to maintain an organism's interaction with its environment.

Animals↗
PubMed2003

Molecular mechanisms of carcinogenesis in gastric cancer.

The catalog of gene alterations in human cancer grows rapidly. Gastric cancer is no exception and displays gene changes in multiple oncogenes, suppressor genes, and DNA repair genes. Clinically relevant molecules whose expression or structure is altered include the plasminogen activator (uPA) and its inhibitor PAI-1 (plasminogen activator inhibitor type 1), the cell-cycle regulator cyclin E, epidermal growth factor (EGF), the apoptosis inhibitor bcl-2, the cell adhesion molecule E-cadherin, and the multifunctional protein beta-catenin. In addition, genetic instability is commonly seen. Gene amplification and protein overexpression of the growth factor receptors c-erbB2 and K-sam may be prognostic factors for intestinal-type and diffuse-type gastric cancer, respectively. The clinical implications of some of the recent findings for diagnosis and therapy are discussed.

Cadherins↗
PubMed1996

Familial breast cancer.

Tremendous progress has been made in recent years in understanding the genetic basis of breast cancer. Epidemiological models have been devised which allow risk estimation for women with one or two relatives with breast cancer. The existence of two autosomal dominant breast cancer susceptibility genes (BRCA1 and BRCA2) have been documented. BRCA1 has been isolated and we are beginning to catalog BRCA1 mutations which appear to be etiologically related to the development of breast cancer in certain families. These BRCA1 mutation studies and the isolation of BRCA2 will allow development of accurate DNA-based testing for the prediction of individual risk in women with a family history of breast cancer. Work by several groups is progressing rapidly on the isolation BRCA2. Ongoing studies investigating the function of BRCA1, and eventually BRCA2, will lead to a better understanding of the role these important genes normally play in preventing the development of breast cancer. Finally, additional breast cancer susceptibility genes are likely to be identified, further expanding the number of tools we have to study, and eventually prevent, breast cancer.

Breast Neoplasms↗
PubMed1995

Evolution of the aminoacyl-tRNA synthetases and the origin of the genetic code.

The aminoacyl-tRNA synthetases exist as two enzyme families which were apparently generated by divergent evolution from two primordial synthetases. The two classes of enzymes exhibit intriguing familial relationships, in that they are distributed nonrandomly within the codon-amino acid matrix of the genetic code. For example, all XCX codons code for amino acids handled by class II synthetases, and all but one of the XUX codons code for amino acids handled by class I synthetases. One interpretation of these patterns is that the synthetases coevolved with the genetic code. The more likely explanation, however, is that the synthetases evolved in the context of an already-established genetic code--a code which developed earlier in an RNA world. The rules which governed the development of the genetic code, and led to certain patterns in the coding catalog between codons and amino acids, would also have governed the subsequent evolution of the synthetases in the context of a fixed code, leading to patterns in synthetase distribution such as those observed. These rules are (1) conservative evolution of amino acid and adapter binding sites and (2) minimization of the disruptive effects on protein structure caused by codon meaning changes.

Amino Acids↗
PubMed1989

Gene of X-chromosomal congenital stationary night blindness is closely linked to DXS7 on Xp.

Congenital stationary night blindness is characterized disturbed or absent night vision that is always present at or shortly after birth and nonprogressive. The X-linked form of the disease (CSNBX; McKusick catalog no. 31050) differs from the autosomal types in that the former is frequently associated with myopia. X-chromosome-specific polymorphic DNA markers were used to carry out linkage analysis in three European families segregating for CSNBX. Close linkage without recombination was found between the disease locus and the anonymous locus DXS7, mapped to Xp11.3, assigning the mutation to the proximal short arm of the X chromosome. Linkage data obtained with markers flanking DXS7 provided further support for this localization of the gene locus. Thus, in addition to retinitis pigmentosa and Norrie disease, CSNBX represents the third well-known hereditary eye disease the locus of which is mapped on the proximal Xp and closely linked to DXS7.

Chromosome Mapping↗
PubMed1989

Regional assignment of the loci for adenylate kinase to 9q32 and for alpha 1-acid glycoprotein to 9q31-q32. A locus for Goltz syndrome in region 9q32-qter?

Normal levels of adenylate kinase (AK-1) and of alpha 1-acid glycoprotein (ORM1) were found in a girl with a deletion 9q32-qter secondary to a maternal translocation (4q35; 9q32), thus excluding these loci from the deleted region. These results, and comparison with other informative data, map the locus for AK-1 to 9q32 and that for ORM1 to region 9q31-q32. The girl has several signs of the Goltz syndrome (Focal dermal hypoplasia), which is listed in the McKusick catalog (no. 30560) as an X-linked dominant condition. Our finding indicates that the locus for Golz syndrome is autosomal and in region 9q32-qter or that there are two such conditions, one autosomal and one X-linked.

Adenylate Kinase↗
PubMed1984

Comparison of one-dimensional IEF patterns for serologically detectable HLA-A and B allotypes.

A new mouse monoclonal antibody (MoAb) 4E, which detects an epitope shared by HLA-B locus antigens, together with the MoAb W6/32, detecting a common HLA, B, C, determinant, and the MoAb4B, detecting HLA-A2 and A28, were used to isolate HLA-A and -B antigens in sequential immunoprecipitation. The HLA antigens obtained from metabolically labeled cell extracts of B-lymphoblastoid cell lines or from phytohemagglutinin (PHA)-activated peripheral blood lymphocytes were compared by one-dimensional isoelectric focusing (1D-IEF). The IEF banding patterns obtained with native HLA antigens segregated in a family with HLA. Neuraminidase treatment of isolated antigens reduced the number of bands to one or two, simplifying the analysis of characteristic patterns. Thus, we have cataloged IEF banding patterns for the majority of the serologically recognized HLA-A and -B allotypes obtained from 57 unrelated American Caucasians. While no HLA-A locus or HLA-B locus specific banding patterns were observed, the HLA-A antigens had, in general, slightly higher pI values than the HLA-B antigens. HLA-C antigens could not be detected in this assay system. The polymorphism detected by IEF banding patterns was as extensive as the serologically detected polymorphism identified by classical HLA serology. Moreover, variants for some HLA allotypes could be detected by this method. In addition to previously recognized A2 variants, new variants were identified for HLA-A1, A26, and Bw44. Each A1 and Bw44 variant was associated with particular haplotypes. The HLA-A2 antigens occurred on 43 HLA haplotypes in the unrelated Caucasian population. Only one of each A2 variants was identified in this population.

Alleles↗
PubMed1985

Evolutionary implications of features of aromatic amino acid biosynthesis in the genus Acinetobacter.

Key enzymes of aromatic amino acid biosynthesis were examined in the genus Acinetobacter. Members of this genus belong to a suprafamilial assemblage of Gram-negative bacteria (denoted Superfamily B) for which a phylogenetic tree based upon oligonucleotide cataloging of 16S rRNA exists. Since the Acinetobacter lineage diverged at an early evolutionary time from other lineages within Superfamily B, an examination of aromatic biosynthesis in members of this genus has supplied important clues for the deduction of major evolutionary events leading to the contemporary aromatic pathways that now exist within Superfamily B. Together with Escherichia coli, Pseudomonas aeruginosa and Xanthomonas campestris, four well-spaced lineages have now been studied in comprehensive detail with respect to comparative enzymological features of aromatic amino acid biosynthesis. A. calcoaceticus and A. lwoffii both possess two chorismate mutase isozymes: one a monofunctional isozyme (chorismate mutase-F), and the other (chorismate mutase-P) a component of a bifunctional P-protein (chorismate mutase-prephenate dehydratase). While both P-protein activities were feedback inhibited by L-phenylalanine, the chorismate mutase-P activity was additionally inhibited by prephenate. Likewise, chorismate mutase-F was product inhibited by prephenate. Two isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase were detected. The major isozyme (greater than 95%) was sensitive to feedback inhibition by L-tyrosine, whereas the minor isozyme was apparently insensitive to allosteric control. Prephenate dehydrogenase and arogenate dehydrogenase activities were both detected, but could not be chromatographically resolved. Available evidence favors the existence of a single dehydrogenase enzyme, exhibiting substrate ambiguity for prephenate and L-arogenate.(ABSTRACT TRUNCATED AT 250 WORDS)

Acinetobacter↗
PubMed1985

Phenylalanine hydroxylase and isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in relationship to the phylogenetic position of Pseudomonas acidovorans (Ps. sp. ATCC 11299a).

The evolution of aromatic amino acid biosynthesis and its regulation is under study in a large assemblage of prokaryotes (Superfamily A) whose phylogenetic arrangement has been constructed on the criterion of oligonucleotide cataloging. One section of this Superfamily consists of a well defined (rRNA homology) cluster denoted as Group III pseudomonads. Pseudomonas acidovorans ATCC 11299a, a Group III member, was chosen for indepth studies of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, the initial regulatory enzyme of aromatic biosynthesis. This strain is of particular interest for evolutionary studies of aromatic metabolism because it possesses phenylalanine hydroxylase, an enzyme whose physiological role and distribution among prokaryotes is largely unknown. Although P. acidovorans ATCC 11299a has been of uncertain identity, we now establish it unambiguously as a species of acidovorans by virtue of its 87% DNA homology with P. acidovorans ATCC 15668 (type strain). This result conformed with enzyme patterning studies which placed ATCC 11299a into pseudomonad Group IIIa, a subgroup containing the acidovorans species. Crude extracts of Group III pseudomonads had previously been shown to share, as a common group characteristic, sensitivity of DAHP synthase to feedback inhibition by either L-tyrosine or L-phenylalanine. Detailed studies with partially purified preparations from strain ATCC 11299a revealed the presence of two distinct regulatory isozymes, DAHP synthase-phe and DAHP synthase-tyr. DAHP synthase-tyr is tightly controlled by L-tyrosine with 50% inhibition of activity being achieved at 4.0 microM effector. DAHP synthase-phe is inhibited 50% by 40 microM L-phenylalanine and exhibits dramatic changes in levels of activity, as well as chromatographic elution patterns, in response to dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1986

Immunocytochemical demonstration of intermediate filament cytoskeleton proteins in human endocrine tissues and (neuro-) endocrine tumours.

The presence and distribution of intermediate filament proteins, such as cytokeratins, vimentin, neurofilament proteins and glial fibrillary acidic protein were assessed immunohistochemically in pituitary adenomas, medullary thyroid carcinomas, endocrine pancreatic tumours, gastric, intestinal and bronchial carcinoids, parathyroid adenomas, pheochromocytomas, paragangliomas and related non-neoplastic tissues. In some cases, immunohistochemical results were correlated with cytoskeletal proteins as analysed by SDS-polyacrylamide gel electrophoresis. Cytokeratin antibodies with broad range of immunoreactivity (i.e. to murine liver cytokeratin component D) reacted with epithelial cells in all non-neoplastic endocrine tissues and related neuroendocrine tumours studied, except for adrenal medulla, pheochromocytoma and paraganglioma, independently of hormone production and biological behaviour. In contrast, antibodies to epidermis-derived cytokeratins failed to stain endocrine tissues and tumours. Paranuclear cytokeratin accumulations were seen in bronchial, gastric, and intestinal carcinoids and seem to be a common feature of neuroendocrine tumours. One- and two-dimensional SDS-polyacrylamide gel electrophoresis of non-neoplastic endocrine tissues and related tumours revealed two major keratin polypeptides corresponding to cytokeratins No. 8 and 18 of the cytokeratin catalog of human cells (Moll et al. 1982). According to this cytokeratin polypeptide composition, endocrine tissues and related tumours conform to the "simple type" of epithelia. Vimentin-related immunoreactivity was restricted to stromal cells and to folliculo-stellate cells in normal pituitary gland, Schwann cells in carcinoids and satellite cells in normal adrenal medulla and in pheochromocytomas. Neurofilament protein- (70 kD)-antibodies only stained nerve fibers in normal tissues and at the periphery of carcinoid tumour cell complexes, and to a variable degree, cells in nontumorous adrenal medulla, pheochromocytomas and paragangliomas. Furthermore, neurofilament reactivity was observed along with cytokeratin expression in two bronchial carcinoids.

Electrophoresis, Polyacrylamide Gel↗
PubMed1982

Mental health needs assessment: beware of false promises.

Needs assessment is considered by experts to be an essential part of mental health planning. Unfortunately, almost anything can pass for a needs assessment. This article briefly examines the methodological limitations of the diverse and unstandardized approaches as well as the conceptual problems associated with needs assessment. The authors suggest that needs assessments could become much more useful if policy-setting and funding bodies would more explicitly state their expectations, if there were greater use of standardized instruments, and if efforts could be directed towards the cataloging of known needs assessment instruments. Further, the state of the art would be advanced if an expert committee could be formed to develop national standards for needs assessments.

Community Mental Health Services↗
PubMed1984

PSRO, quality review and mental health--a political and practical guide.

Quality assessment of mental health care is distinguished by it defensive character. The field has been sensitive to the criticisms of outside agencies and groups to the point of studying the quality of care it provides far more than other health providers have done. The result has been productive leadership in matters of practical evaluation. The author details the impact of PSRO, the JCAH, DRGS, and other formal review mechanisms upon the quality assessment process. He catalogs a shift in emphasis to take into account organizational dynamics and concludes that the JCAH seems to be propelling alcohol, drug, and mental health treatment programs into creative new directions.

Alcoholism↗
PubMed1992

Manometric patterns using esophageal body and lower sphincter characteristics. Findings in 1013 patients.

In order to determine the actual spectrum of abnormal esophageal motility, manometric patterns in 1013 consecutive tracings were established using a classification method that employs esophageal body and lower esophageal sphincter (LES) characteristics. Peristaltic performance and contraction wave parameters were measured in the esophageal body; basal pressure and relaxation were included for the LES. Nine hundred thirty (92%) of the tracings could be completely classified, and 33 different patterns were observed (915 occurring at a rate greater than 1%). Abnormalities were most common in contraction wave parameters (661 tracings, 65%), and least common in LES relaxation (105 tracings, 10%). Patterns most typical of achalasia and diffuse esophageal spasm were found in 6.4% and 5.0% of tracings, respectively. Statistical analysis of the patterns demonstrated that significant bidirectional predictive associations between categories were restricted to features representing pathology-based motor disorders (ie, achalasia and "scleroderma-esophagus"). This systematic classification method is capable of recognizing and cataloging common findings of motor dysfunction in the esophageal body and LES as well as uncommon patterns representing traditional motility disorders. Our findings provide reference data for clinical esophageal manometry.

Esophageal Motility Disorders↗
PubMed1996

MR of the pituitary in patients with Prader-Willi syndrome: size determination and imaging findings.

Prader-Willi syndrome (PWS) is an unusual genetic disorder characterized by short stature, obesity, hypogonadism, hypotonia, cognitive impairment, and dysmorphic facies. There is an interstitial deletion of the proximal long arm of chromosome 15 in about 70 % of patients. Some of these clinical features suggest a central hypothalamic/pituitary dysfunction, and recent investigations have demonstrated a marked impairment in spontaneous growth hormone (GH) secretion. We studied 15 GH-deficient PWS patients by magnetic resonance imaging (MRI) to determine whether there was a diminution in the gross morphological size of the anterior pituitary gland, the site of GH synthesis. We also set out to catalog the pertinent imaging findings in this patient population. Our results indicate that this is the first report documenting pituitary size by MRI in PWS patients. No statistically significant difference was found in the height of the anterior pituitary gland in PWS patients compared with either normal children or children with isolated GH deficiency. An interesting imaging finding is that three of 15 patients (20 %) demonstrated complete absence of the posterior pituitary bright spot (PPBS), and a fourth patient demonstrated a small PPBS. These observations reflect an objective physiologic disturbance in the hypothalamus. The clinical and radiologic implications of these findings are discussed.

Adolescent↗