Purpose Male infertility is a complex health condition. To our knowledge there are no molecular biomarkers of male infertility. Sperm RNA is a potential biomarker for detecting sperm abnormalities ...and viability at infertility clinics. However, RNA use is hindered by its inconsistent quantity, quality, multiple cell types in semen and condensed sperm structure. Materials and Methods We tested the usefulness of high quality RNA isolated from mature sperm and whole semen by our protocol, which reduces RNA degradation by maintaining semen and protocol components at 37C and decreasing processing time. We isolated RNA from 83 whole semen samples, 18 samples of motile sperm prepared by the swim-up protocol and 18 of sperm prepared by the standard Percoll gradient method. Results Electrophoretic and spectral analysis of RNA revealed high quality 18S and 28S rRNAs in 71 of 83 whole semen samples (86%) and 15 of 18 mature sperm swim-up samples (83%). However, high quality RNA was isolated from only 7 of 18 Percoll gradient sperm samples (39%). Interestingly quantitative reverse transcriptase-polymerase chain reaction analysis of 4 somatic and 10 germ cell markers showed that whole semen and swim-up samples had similar RNA profiles. RNA sequencing revealed that most encoded proteins were involved in mature sperm function, regulation of DNA replication, transcription, translation, cell cycle and embryo development. Conclusions We believe that semen and sperm specific RNAs are highly informative biomarkers for germ cell stages and somatic cell contribution. Therefore, these RNAs could be valuable diagnostic indicators of sperm survival, fertilization and early embryogenesis, and could serve as a predictor of the in vitro fertilization prognosis.
Mammary gland development culminates in lactation and is orchestrated by numerous stimuli and signaling pathways. The Src family of nonreceptor tyrosine kinases plays a pivotal role in cell ...signaling. In order to determine if Src plays a role in mammary gland development we have examined mammary gland development and function during pregnancy and lactation in mice in which expression of Src has been eliminated.
We have characterized a lactation defect in the Src-/- mice which results in the death of over 80% of the litters nursed by Src-/- dams. Mammary gland development during pregnancy appears normal in these mice; however secretory activation does not seem to occur. Serum prolactin levels are normal in Src-/- mice compared to wildtype controls. Expression of the prolactin receptor at both the RNA and protein level was decreased in Src-/- mice following the transition from pregnancy to lactation, as was phosphorylation of STAT5 and expression of milk protein genes. These results suggest that secretory activation, which occurs following parturition, does not occur completely in Src-/- mice. Failed secretory activation results in precocious involution in the mammary glands of Src-/- even when pups were suckling. Involution was accelerated following pup withdrawal perhaps as a result of incomplete secretory activation. In vitro differentiation of mammary epithelial cells from Src-/- mice resulted in diminished production of milk proteins compared to the amount of milk proteins produced by Src+/+ cells, indicating a direct role for Src in regulating the transcription/translation of milk protein genes in mammary epithelial cells.
Src is an essential signaling modulator in mammary gland development as Src-/- mice exhibit a block in secretory activation that results in lactation failure and precocious involution. Src appears to be required for increased expression of the prolactin receptor and successful downstream signaling, and alveolar cell organization.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Subunit interaction in the formation of active acyl-coenzyme A:isopenicillin N acyltransferase (AT) has been investigated. Various AT derivatives were produced from altered Penicillium chrysogenum ...penDE genes placed in Escherichia coli expression systems. The regions of penDE encoding the alpha (11 kDa) and beta (29 kDa) AT subunits were separated at the DNA level by linker insertion at the region encoding Gly(102)/CYS(103). Synthesis of AT from the resulting two-cistron mRNA resulted in active alpha,beta-heterodimeric recombinant AT (reAT), containing subunits of 11 and 29 kDa (similar to wild-type AT). Complete separation of the alpha and beta subunits was performed by placing the region of penDE encoding each subunit on different plasmids. Production of either subunit in the absence of the other did not form active reAT. However, cotransformation of E. coli with two plasmids, each encoding a different AT subunit, produced reAT having acyl-coenzyme A:6-aminopenicillanic acid (acyl-CoA:6-APA) AT activity. Mutation of penDE replacing Thr(105) with Asn resulted in inactive and uncleaved reAT. Coexpression of this mutant penDE with a penDE derivative encoding the beta subunit in E. coli produced acyl-CoA:6-APA AT activity. These results suggest that the formation of reAT involves cooperative folding events between the subunits. In vitro transcription/translation was used to determine the origin of the AT hydrolase activity that cleaves the 40-kDa precursor polypeptide. The appearance of a 29-kDa protein (and presumably the corresponding 11-kDa protein, although not observable) from the 40-kDa in vitro translated protein provides further evidence that AT hydrolysis is an autocatalytic event.
Sequence Correction Morris, S. W.; Kirstein, M. N.; Valentine, M. B. ...
Science (American Association for the Advancement of Science),
01/1995, Letnik:
267, Številka:
5196
Journal Article
Recenzirano
We wish to note a correction of the
NPM-ALK
sequence (GenBank accession number U04946) described in our report "Fusion of a kinase gene,
ALK
, to a nucleolar protein gene,
NPM
, in non-Hodgkin's ...lymphoma" (4 March 1994, p. 1281). After re-examination of our sequencing data, we identified an erroneous omission of two nucleotides in the codon of NPM-ALK amino acid residue 495 that was made because of a reading error. The frame-shift produced by this error led us to assign a premature termination codon, resulting in a predicted NPM-ALK protein of 525 amino acids. The correct
NPM-ALK
nucleotide sequence, now entered in GenBank, encodes a protein of 679 amino acids with a predicted molecular weight of 75.3 kilodaltons. A polypeptide with this relative mobility, generated by in vitro transcription/translation of the full-length
NPM-ALK
complementary DNA, co-migrates with the NPM-ALK protein immunoprecipitated from the t(2;5)-containing lymphoma cell lines SU-DHL-1, SUP-M2, and UCONN-L2.
To our knowledge, all other data reported in our paper are correct. We regret any inconvenience or confusion that this sequencing error may have caused our scientific colleagues.
With the previously obtained rat liver serine dehydratase cDNA (SDH2; Ogawa, H., Miller, D.A., Dunn, T., Su, Y., Burcham,
J. M., Peraino, C., Fujioka M., Babcock, K., and Pitot, H. C. (1988) Proc. ...Natl. Acad. Sci. U.S. A. 85, 5809-5813) as a probe,
we isolated a different species of cDNA (SDH3) from the same cDNA library from which SDH2 was obtained. Nucleotide sequence
analysis has indicated that SDH3 has an open reading frame which encodes 327 amino acid residues and which is identical to
that of the cDNA obtained by Noda et al. (Noda, C., Ito, K., Nakamura, T., and Ichihara, A., (1988) FEBS Lett. 234, 331-335).
Primer extension analysis and RNase protection mapping clarified that the SDH3 mRNA was the major mRNA for serine dehydratase
in the liver, and its transcription begins with a T residue located 23 nucleotides down-stream of a TATA-like box. In vitro
transcription/translation experiment demonstrated that SDH3 encoded a polypeptide of 35 kDa, a size in agreement with that
of the subunit of the purified protein, whereas SDH2, despite having a size larger than SDH3, produced a peptide of much smaller
size that reacted with anti-serine dehydratase IgG. SDH2 was found to have a stop codon early in the sequence and is predicted
to encode a polypeptide of 8.9 kDa. Also, SDH2 has a 5'-noncoding sequence different from that of SDH3. These results indicate
that alternative transcription initiation and different modes of splicing of the primary transcripts of rat serine dehydratase
gene result in the formation of two species of mRNA, of which only one is translated into the mature serine dehydratase protein.
Expression of the bcl-2 gene becomes deregulated in many non-Hodgkin lymphomas as the result of t(14;18) chromosomal translocations. Because bcl-2 regulates the survival of cells, and because its ...over-expression is associated with cellular resistance to killing by chemotherapeutic drugs and gamma-irradiation, this gene and its mRNA and protein products represent ideal targets for designing novel therapeutic strategies for the treatment of cancer. Here we describe the effects of an 18-mer phosphodiester oligonucleotide that is complementary to the first 6 codons of the bcl-2 mRNA's open reading frame. When tested for inhibition of in vitro protein synthesis using RNAse-H-supplemented reticulocyte lysates and RNA prepared by in vitro transcription of a human bcl-2 cDNA, the bcl-2 antisense (AS) oligomer completely abolished Bcl-2 protein production at 10 microM, but had no effect on the in vitro translation of a chicken bcl-2 RNA that contained three mismatches relative to the oligomer binding site on the human bcl-2 RNA. A control 18-mer having the same base composition as the AS oligomer but with scrambled order (SC) was not inhibitory. Addition of AS and SC oligomers to cultures of a NIH-3T3 fibroblast cell line that had been stably infected with a recombinant retrovirus containing the same human bcl-2 cDNA used for in vitro transcription/translation experiments revealed concentration-dependent reductions in the relative levels of the 26-kD human Bcl-2 protein (as determined by immunoblotting) by the AS but not by the SC oligomer. Similar results were obtained when AS and SC oligomers were applied to a t(14;18)-containing lymphoma cell line SU-DHL-4 that was cultured in low-serum media. When used at 200 microM, the bcl-2 AS oligomer produced 84-95% reductions in Bcl-2 protein levels in SU-DHL-4 cells but had relatively little effect on the levels of other mitochondrial control proteins, suggesting that the inhibitory effects were specific. Treatment of SU-DHL-4 cells with AS oligomer lead to essentially complete loss of bcl-2 mRNA from cells within 1 day of addition to cultures, but presumably because of the long half-life of the Bcl-2 protein (approximately 14 h), commensurate reductions in Bcl-2 protein levels did not occur until 3 days.
We have been faced with a 40 year long challenge: how to establish tools that can be applied in the treatment of brain tumor - glioblastoma (100% fatal) - using our knowledge of evolution, chemistry ...of proteins, genetics, molecular biology and immunology. An efficient strategy targeting growth factor IGF-I, present in tumor development, was established by construction of vectors expressing either IGF-I antisense RNA or IGF-I RNA forming RNA-DNA triple helix. The vectors introduced in the cancer cells in vitro, enable to completely stop the synthesis of IGF-I: on translation or transcription level, respectively. When injected in vivo, these cells induce an immune anti-tumor effect (CD8+) accompanied by increase of the median survival of patients. The first thesis in Colombia describing the used technology, was presented in Distrital University in February 2016.
Unos de los retos científicos de los últimos 40 años, ha sido la búsqueda de la herramienta para el tratamiento del tumor cerebral, el glioblastoma, mortales en el 100% de los casos, utilizando nuestro conocimiento de la evolución, la química de las proteínas, la genética, la biología molecular y la inmunología. Una estrategia eficiente enfocada hacia el factor de crecimiento IGF-I presente en el desarrollo tumoral, fue establecida mediante la construcción de vectores expresando el IGF-I antisentido ARN o el IGF-I ARN formando ARN-ADN triple hélice. Estos vectores introducidos en las células cancerosas in vitro, permiten detener por completo la síntesis de IGF-I en la traducción o a nivel de la transcripción respectivamente. Mientras la inyección in vivo, inducen efecto antitumoral inmune (TCD8+) acompañado de aumento de la supervivencia media de los pacientes. La primera tesis en Colombia que describe la tecnología utilizada, fue presentada en la Universidad Distrital, en febrero de 2016.
Los virus de plantas para extender la infección necesitan, en su movimiento célula-célula, atravesar la barrera que supone la pared celular, para lo cual aprovechan los plasmodésmos (PD). Éstos son ...aperturas en la pared celular atravesadas por un canal membranoso (desmotúbulo) que permiten el intercambio de agua, nutrientes y moléculas de señalización. En el caso de los virus de RNA de polaridad positiva la entrada de la partícula viral en la planta provoca el desensamblaje de la cubierta. Proceso ligado al inicio de la transcripción de las primeras proteínas virales, implicadas en la replicación del genoma viral. A continuación se sintetizan el resto de proteínas del virus entre ellas las encargadas de transportar el RNA viral de una célula a la célula adyacente, conocidas como proteínas de movimiento (MP). Estas proteínas son imprescindibles para el transporte del genoma del virus, en un primer lugar al PD (empleando la red de membranas derivadas del retículo endoplasmático (ER)) y a continuación a través de éstos; en este caso necesariamente en intensa interacción con el desmotúbulo. Los virus de plantas se agrupan en tres grandes categorías según el número de MP que presenten, así encontramos aquellos que presentan una única MP de gran tamaño (la superfamilia 30k), otro gran grupo conocido como DGB (double gene block) con dos pequeñas MP y por último el TGB (triple gene block) con tres MP. La relación entre las membranas de la planta y las MP es imprescindible para el transporte del genoma viral pero únicamente ha sido estudiada en profundidad en el caso del TGB. Es por esto que en la presente tesis se planteó el estudio del tipo de interacción establecida entre la membrana y las MP del los virus MNSV y TCV ambos del DGB y PNRSV y TMV (pertenecientes a la superfamilia 30k). Para lo cual se emplearon métodos bioquímicos de transcripción y traducción in vitro en presencia de microsomas y diferentes técnicas biofísicas como dicroísmo circular o la balanza de Langmuir así como ensayos in vivo tanto en E.coli como en N.bentamiana. El MNSV tiene dos MP, una soluble y capaz de unir RNA (p7A), la otra p7B presenta una única región hidrofóbica (RH) entre los aminoácidos 13 y 32 identificada por los programas de predicción empleados como un fragmento transmembrana (fTM). Dicha región es capaz de insertarse en membranas del ER. Es además capaz de dirigir e insertar la proteína en la membrana de manera cotraduccional. p7B adopta tanto en membranas del ER como en E.coli una topología N-t citosólico/C-t extracelular. La MP p9 del TCV al igual que p7B se inserta de manera cotraduccional en membranas del ER, donde adopta una topología N-t citosólico/C-t luminal determinada por su único fTM (residuos 3-20).La MP del PNRSV, p30 presenta una RH incapaz de insertarse en la membrana. La proteína si se asocia a las membranas aunque de manera periférica no de manera integral. Su dominio hidrofóbico (aa 89-110) es probablemente responsable de esta interacción y juega un papel fundamental en la capacidad de la proteína para transportar el RNA viral de una célula a otra. La prolina 96 (en la RH) además de determinar la estructura de este dominio es imprescindible para la correcta función de la proteína. La MP del TMV p30, ha sido considerada hasta la fecha como una proteína integral de membrana. Los estudios realizados en esta tesis demuestran que si bien si se asocia a la bicapa lipídica no lo hace de manera integral sino más bien periféricamente, análogamente a p32.
The cell-to-cell movement of RNA plant virus require the function of the so-called movement proteins (MP). These proteins interact with the plasmodesmata, an aperture in the plant cell wall crossed by a membranous channel formed by prolongations of the Endoplasmatic Reticulum (ER), to facilitate the passage of the viral RNA from one cell to the next one. The interaction of the MP with the ER has been reported for several MP in different stages of the infectious process, but the nature of this association has only been fully studied in few cases. To gain further knowledge of the infection process we have examined the type of association of three different MP, p7B (MNSV), p9 (TCV), p30 (TMV) and p32 (PNRSV) with membranes derived from the ER. In this study we have use a broad range of biochemical techniques, from in vitro transcription and translation to circular dichroism, monolayer studies and membrane isolation from plant tissue. The experimental approach was supplemented with a computer-assisted analysis of the MP sequences. In this work we have found that p7B inserts into the membrane in a co-translational manner using its hydrophobic region (aminoacids 13-32) as a signal sequence and as a stop transfer sequence. The N-t of the protein faces the cytosol while the C-t its located within the lumen of the ER. p9 from TCV is also a Type II integral membrane protein with just one transmembrane region (aminoacids 3-20). The MP from PNRSV has one hydrophobic region (aminoacids 89-110), which it is not capable of being inserted into microsomal membranes by the translocon machinery. In despite of not being transmembrane this segment seems to be essential for the membrane association of the MP and for the cell-to-cell RNA transport shown by p32. Our results indicate that p30 is not integral membrane proteins but a membrane associated protein.It seems that the association of MP to membranes it is necessary for the infectious process, although plant virus have evolve different strategies to accomplish this task.
PDF
