RESUMO
In low activity gamma spectrometric measurements of large, dense samples, the bulk sample material shields the HPGe crystal from external background sources. If not accounted for in studies that utilise background-subtraction methods, this effect may result in systematic errors in the sample activity and detection limit estimation. We introduce a Monte Carlo based method to minimise the impact of this effect on sample gamma spectra. It is validated using simulated detector backgrounds and applied to a measurement of low-activity [Formula: see text] . One main prerequisite for the correct application of this method is to know in advance the nuclides which contribute to the detector background spectrum and their spatial distribution. With a thorough understanding of the detector backgrounds, the method improves the accuracy of sensitive low-background measurements of low-activity samples. Even without knowing the background sources and their distribution, conservative results may still be presented that account for the potential systematic errors introduced by this background shielding effect.
Assuntos
Espectrometria gama , Raios gama , Método de Monte Carlo , Espectrometria gama/métodosRESUMO
Type 2 cystatins comprise a class of cysteine peptidase inhibitor presumed to mediate protective functions at various locations, including the oral cavity. Seven cystatin genes are clustered within a 300-kb region of human 20p11.2. "Salivary" cystatins, encoded by CST1, 2, 4, and 5, are present in saliva at significant levels but have also been reported in other secretions, such as tears, suggesting that during their evolution, these genes have acquired mechanisms directing differential tissue-specific expression. However, their patterns of expression, which might also provide additional clues to their individual functions, have not been determined. Gene-specific RNase protection assays were used to examine the qualitative and quantitative distribution of expression of these seven genes within a collection of 23 adult human tissues. The CST3 gene, encoding cystatin C, was expressed at modest levels in all tissues examined. The presumptive pseudogenes CSTP1 and CSTP2 were not expressed at detectable levels in any tissue. The CST1, 2, 4, and 5 genes were expressed in differential, tissue-specific patterns. Expression of CST2 and CST5 was restricted to the submandibular and parotid glands, while CST1 and CST4 were expressed in these tissues and in the lacrimal gland. Immunohistochemistry studies localized expression to the serous-type secretory end pieces. Coexpression of CST1 and CST4 was also observed in the epithelial lining of the gallbladder and seminal vesicle. The CST1 product was detected in the tracheal glands and CST4 in the kidney and prostate. Despite their different adult patterns of expression, analysis of CST1, 2, 4, and 5 mRNA levels in infant submandibular glands demonstrated a coordinate upregulation of expression of between 3.5 and 9 months of age. The patterns of cystatin gene expression are consistent with several proposed oral functions of the salivary cystatins but also suggest they are important in other locations and that, despite their close sequence similarity, they are individually specialized.
Assuntos
Cistatinas/genética , Inibidores de Cisteína Proteinase/genética , Expressão Gênica , Glândula Submandibular/metabolismo , Adolescente , Adulto , Idoso , Sequência de Bases , Criança , Pré-Escolar , Cistatinas/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Lactente , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sondas RNA , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Homologia de Sequência do Ácido Nucleico , Distribuição TecidualRESUMO
An abundant 1.05 kb human lacrimal gland mRNA has been characterized by cDNA cloning. It encodes a predicted 180 residue, 20546 Da secreted protein, with a charge of +11 at ph 7 and 24.5% proline, designated as Basic Proline-rich Lacrimal Protein (BPLP), Southern blot analysis is consistent with a single BPLP gene. BPLP lacks any distinct repetitive structure, and is unrelated to the salivary proline-rich protein super-family. The pre-proprotein shows modest overall similarity to a superfamily comprising human PRPb, the mouse MSG proteins, and rat VCS-alpha 1, VCS-beta 1 and submandibular apomucin. BPLP also contains a domain with similarity to the Zp2 protein domain found in several otherwise unrelated proteins. Northern blot analysis indicated that the BPLP gene is also expressed at modest levels in the human submandibular gland, and in situ hybridization demonstrated expression of BPLP in the secretory endpieces of the human lacrimal gland. The BPLP cDNA clone defines a new human tear protein, and should provide a useful phenotypic marker of differentiation in in vitro studies of lacrimal gland function.
Assuntos
DNA Complementar/genética , Proteínas do Olho/genética , Aparelho Lacrimal/metabolismo , RNA Mensageiro/genética , Lágrimas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Ratos , Homologia de Sequência de AminoácidosRESUMO
PURPOSE: To examine the existence of novel protein products of the human lacrimal gland. METHODS: cDNA clones corresponding to a highly abundant human lacrimal gland mRNA were isolated and sequenced. Tissue distribution of expression was studied by Northern blot analysis, RNase protection analysis, and in situ hybridization. RESULTS: A highly abundant 600-base mRNA was identified, and corresponding cDNA clones were isolated. The mRNA has a 134-residue open reading frame encoding a secreted protein of 13458 Da. This protein shows 45.5% similarity to human salivary acidic proline-rich protein PRP 1 and has a similar domain structure, but, unlike other members of the proline-rich protein family, it lacks a conserved repetitive domain. The lacrimal proline-rich protein is encoded by a single gene, designated as LPRP. Expression of LPRP was also detected in the human submandibular, von Ebners, sublingual, and parotid glands. LPRP was expressed in the acinar cells of the lacrimal gland, and in the submandibular gland expression of the LPRP and PRP 1 genes was localized to the serous acini and demilunes. CONCLUSIONS: The human lacrimal gland produces a previously unknown member of the proline-rich protein family. By analogy with other proline-rich proteins, this LPRP most likely mediates protective functions in the eye, such as modulation of the microflora. In contrast to other proline-rich protein genes, LPRP is expressed in the lacrimal acinar cells, and other anterior exocrine glands. LPRP should be a useful marker for human lacrimal gland acinar cell function in vitro.
Assuntos
Proteínas do Olho/biossíntese , Aparelho Lacrimal/metabolismo , Biossíntese Peptídica , Prolina/biossíntese , RNA Mensageiro/biossíntese , Adulto , Idoso , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Clonagem Molecular , Feminino , Humanos , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Prolina/química , Domínios Proteicos Ricos em Prolina , RNA/análise , RNA/química , RNA Mensageiro/análise , Sequências Repetitivas de Ácido Nucleico , Glândulas Salivares/citologia , Glândulas Salivares/metabolismo , Proteínas Salivares Ricas em Prolina , Proteínas e Peptídeos Salivares/biossínteseRESUMO
The human type 2 cystatins are encoded by a multigene family of eight to nine members. Five functional genes (CST1-CST5) and two pseudogenes (CSTP1 and CSTP2) have previously been cloned. We have developed a panel of short genomic fragments that serve as gene-specific probes for these seven genes under hybridization conditions used for Southern blots or fluorescence in situ hybridization (FISH). Analysis of BssHII-digested genomic DNA using these probes was consistent with association of all seven genes with a 905-kb fragment, suggesting physical clustering at a single site. The cluster was assigned to chromosome 20 by screening a somatic cell hybrid panel with an STS for CST2. This assignment was confirmed for each gene by FISH using repeat-free gene-specific genomic probes ranging in size from 2.3 to 0.8 kb. All the loci mapped to 20p11.2.
Assuntos
Cromossomos Humanos Par 20 , Cistatinas/genética , Animais , Sequência de Bases , Células CHO , Mapeamento Cromossômico , Cricetinae , Primers do DNA , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Dados de Sequência MolecularRESUMO
The family of type 2 cystatin proteins are a class of cysteine proteinase inhibitors found in a variety of human fluids and secretions, where they appear to provide protective functions. To establish the size of the human gene family encoding these proteins, we isolated cosmid and lambda genomic clones. Restriction mapping, partial sequence analysis, and hybridization studies identified a total of seven distinct genes, six of which correspond to known genes and proteins. Sequence analysis showed that the seventh gene, CSTP2, is an apparent pseudogene carrying a nonsense mutation in exon 1 distinct from that in CSTP1. Southern blots of genomic DNA probed with gene-specific probes accounted for all but one or two sets of fragments containing exon 1, and one or two sets of fragments containing exon 3, indicating that the human type 2 cystatin gene family consists of eight or nine members. Southern blot analysis of large restriction fragments using these gene-specific probes indicates that all seven of the cloned type 2 cystatin genes are clustered at a single locus on human chromosome 20. This locus is no larger than about 910 kb, and possibly as small as 365 kb. We designate this as the CST locus and suggest a numbering system for the cystatin genes.
Assuntos
Cromossomos Humanos Par 20 , Cistatinas/genética , Família Multigênica , Sequência de Aminoácidos , Bacteriófago lambda , Sequência de Bases , Southern Blotting , Clonagem Molecular , Cosmídeos , DNA , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Mapeamento por RestriçãoRESUMO
Humans carry one gene encoding cystatin C and six to eight genes with homology to an S-like cystatin hybridization probe. However, the precise composition and organization of the cystatin gene family remains to be established. Further, the pattern of tissue-specific expression has not been fully defined. We have previously shown that the type 2 cystatin genes are clustered together in a ca. 270 kb region (the CST locus). To determine the structure of this region, we have sought to clone the entire CST locus. Our approach has been to isolate cosmid and lambda genomic clones carrying cystatin genes and then to use "walk" probes derived from the end regions of these clones to identify other clones, which extend them. To date, we have obtained over 320 kb of distinct sequences. Based on restriction maps, sequencing, and hybridization analyses, we have identified eight apparently nonallelic copies of cystatin genes. These include one gene for cystatin C, four closely related genes encoding S-like cystatins, and three genes encoding relatively divergent sequences. Complete assembly of these clones into an unambiguous contiguous sequence is hampered by the presence of flanking locus-specific repetitive-like sequences. RNase protection assays used to characterize the tissue-specific patterns of expression showed that cystatin C is expressed at modest, comparable levels in all tissues examined, whereas expression of the CST 1 gene, encoding cystatin SA-I, was found to be restricted to a small subset of tissues, with the highest level in the submandibular gland.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 20 , Cistatinas/genética , Expressão Gênica , Passeio de Cromossomo , Clonagem Molecular , Sondas de DNA , Genoma Humano , Biblioteca Genômica , Humanos , RNA/análise , Mapeamento por Restrição , Análise de Sequência de DNARESUMO
Cell production and cell deaths were determined in larval Rana pipiens both in control tecta and in tecta following unilateral eyeball removal in embryos and larvae. Such enucleations produce significantly reduced rates of cell division in the contralateral tecta for virtually the entire larval period (confirming studies with enucleation almost exclusively performed in embryos--Kollros: J. Exp. Zool. 123:153-187, '53, and J. Comp. Neurol. 205:171-178, '82). Significant numbers of cell deaths in all nonependymal tectal cell layers were also observed. Control cell division rates peak at stage X, while cell death peaks are reached in stages XIII-XX. Overall, about 10(6) nonependymal cells are produced in control tecta, and about 350,000 of them die by the end of metamorphosis. Control of cell numbers following enucleation is shown to depend mainly on reductions in cell division rates when the operation occurs early in development and mainly on increases in cell death rates when the operation occurs late in larval life. Such increases in death rates are invariably present within 1 day of the operation whereas the reduced division rates ordinarily require several more days to be seen. The modified rates, both of cell divisions and cell death, are limited to tectal areas to which optic nerve fibers have already extended. Maps of the positions of tectal cell divisions in many larval stages provide the basis for modifying the current dogma that tectal formation occurs as a series of newly formed mediocaudal wedges pushing previously produced wedges rostrolaterad. All such "old" wedges receive substantial cell additions for many stages, with the rate of addition decreasing rostrad earlier than caudad.
Assuntos
Rana pipiens/crescimento & desenvolvimento , Colículos Superiores/crescimento & desenvolvimento , Animais , Contagem de Células , Divisão Celular , Sobrevivência Celular , Larva , Nervo Óptico/crescimento & desenvolvimento , Rana pipiens/anatomia & histologia , Colículos Superiores/citologiaRESUMO
Positions, numbers, and cell and nuclear sizes of mesencephalic fifth nucleus (M-V) cells were determined in Xenopus laevis larvae in stages 47 through the end of metamorphosis at stage 66. M-V cells may be found at every tectal level, from the rostralmost section almost to the caudal pole, and in the anterior medullary velum. A large majority of the cells lie between 15 and 65% caudad of the rostral tip of the tectum. At anterior and middle tectal levels the cells lie lateral to but mainly above the optic ventricle. At posterior levels, to which the ventricle does not extend, a few cells may be seen at middle and lower tectal levels, as if in transition to the anterior medullary velum. At stage 47 fewer than ten cells are seen in each animal. The numbers rise to 20-40 by stage 50, and are uniformly above 100 after stage 51. Initially many M-V cells were small, i.e., 6-7 microns in diameter, but grew to a mean diameter of about 19 microns at stage 59, with a maximum value of 29 microns. A single individual at stage 57 had 581 cells. The peak of mean cell numbers, 387, occurred at stage 59, which was also the stage with the highest mean values for nuclear and cell sizes. Pyknotic M-V cells at low frequency were seen at stages 55 and 57, and at all stages thereafter. Cell death frequency peaked at stage 62, but continued through stage 66. By stage 66 mean cell numbers had been reduced to about 240, indicating survival of about 60% of cells present at stage 59.