Biomedical subjects

C Risco

Publications and source records attributed to C Risco.

At least 19 recordsLinked to original sources

PubMed2002

Effects of whole cottonseed diet and recombinant bovine somatotropin on ovarian follicles in lactating dairy cows.

The effects of whole cottonseed (WCS) in the diet and the administration of bovine somatotropin (bST) on ovarian follicular dynamics and plasma progesterone (P4) concentrations were examined in cows during a period of synchronized follicular growth. Lactating Holstein cows (n = 28) were randomly assigned to treatments in a 2 x 2 factorial arrangement. Diets consisted of WCS (15% of dry matter) or no WCS, and bST at a dose of 0 or 208 mg/14 d. Dietary treatments began within 24 h of calving and bST treatments began within 7 d postpartum. Cows received GnRH at 65 +/- 3 d postpartum (d 0), PGF2alpha, (d 7), a second dose of GnRH (d 9), and were inseminated 16 h later (d 10). Ovarian changes were monitored daily by ultrasonography from d 0 to 9. On d 9,93% of cows had a preovulatory follicle and 86% ovulated. For Class 2 (6 to 9 mm) follicles, a diet x bST interaction was detected, with bST stimulating Class 2 follicles in cows fed WCS, but not in cows on the control diet. Neither diet nor bST affected numbers of Class 1 (2 to 5 mm) or Class 3 (> or = 10 mm) follicles or sizes of the subordinate and dominant follicles. During the luteal phase of the cycle, lactating cows fed WCS tended to have elevated concentrations of plasma P4, whereas bST was without effect. Plasma concentrations of high-density lipoprotein cholesterol were increased in cows fed WCS. Number and diameter of corpora lutea did not differ among treatments.

Animal Feed↗

PubMed2001

The A17L gene product of vaccinia virus is exposed on the surface of IMV.

The p21 membrane protein of vaccinia virus (VV), encoded by the A17L gene, has been reported to localize on the inner of the two membranes of the intracellular mature virus (IMV). It has also been shown that p21 acts as a membrane anchor for the externally located fusion protein p14 (A27L gene). Since p14 is located on the surface of IMVs, it is hard to envision that p21 should be located only on the inner membrane. Our results from (i) immunoelectron microscopy, (ii) biotinylation, and (iii) protease treatment of purified IMVs showed that the N-terminus of p21 is exposed on the surface of virus particles, while the C-terminus is embedded in the membrane. Mono-specific antibodies to the N-terminus of p21 neutralize infection of VV while antibodies to the C-terminal domain do not. We suggest that p21 molecules are located both in the inner and in the outer membrane of IMV.

Animals↗

PubMed2001

Association between exposure to Neospora caninum and milk production in dairy cows.

OBJECTIVE: To determine association between exposure to Neospora caninum and milk production in dairy cows. DESIGN: Prospective observational study. ANIMALS: 565 Holstein cows. PROCEDURE: Cows were classified as seropositive or seronegative to N. caninum within 7 days after calving by use of a kinetic ELISA. Milk production was compared between seropositive and seronegative cows. RESULTS: On the basis of 305-day mature equivalent milk production data, seropositive cows produced less milk (2.8 lb/cow per day) than did seronegative cows. In addition, analysis of results throughout the first 300 days of lactation revealed that after adjusting for effects of lactation number, calving season, clinical mastitis, and lameness, milk weight of seropositive cows was 2.5 lb/cow per day less than that of seronegative cows. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure to N. caninum was associated with a 3 to 4% decrease in milk production. A decrease in milk production of 800 lb/cow for a typical 305-day lactation represents a loss of $128/cow.

Animals↗

PubMed2001

Elemental maps from EFTEM images using two different background subtraction models.

Acquisition of a great number of energy-filtered images in a TEM (EFTEM) around the characteristic signal with a low energy-selecting slit allows display of the electron energy loss (EEL)-spectrum of regions of interest (ROIs) of a sample. These EEL-spectra can be submitted to the different treatments already in use for electron energy loss spectroscopy (EELS). In particular, it is possible to fit the experimental background with different mathematical models, using images acquired below and above a characteristic ionization edge. After this fitting, elemental maps can be computed by subtraction of the extrapolated/interpolated background from the characteristic images. In this work, we compared two mathematical models for background fitting-the Egerton power law and the log-polynomial law. We studied the low-energy region (40-150 eV) and a higher-energy region (350-600 eV) with the aid of software for interactive processing of EFTEM image series that we developed. The analyzed elements were the constitutive elements: iron, phosphorus, nitrogen, and oxygen in several biological materials. Two analytical TEMs, one equipped with a post-column and the other with an in-column spectrometer, were used. Our experimental results confirm that the power law is very sensitive to the value of the energy loss of the pre-edge images when the background is computed by extrapolation. The log-polynomial model is less sensitive than the power law model to the value of the energy loss of the pre-edge images in the low energy region. For the oxygen K edge at 535 eV, it gives the best fit when it is combined with the interpolation method. The use of programs that facilitate the handling of EFTEM image series, and the controlled calculation of the background under the characteristic images, represent a step forward in the generation of elemental maps.

Animals↗

PubMed2001

Multiple regulatory mechanisms act on the 5' untranslated region of the S-layer gene from Thermus thermophilus HB8.

The role of the 5' untranslated region (5'UTR) of the S-layer gene from Thermus thermophilus was analyzed through the isolation of Delta 5'UTR mutants. In these mutants the half-life of splA mRNA was strongly reduced and slpA transcription was no longer subjected to growth phase-dependent repression. Overproduction and detachment of the external envelopes of the mutants were observed in stationary phase.

5' Untranslated Regions↗

PubMed2001

The major core protein P4a (A10L gene) of vaccinia virus is essential for correct assembly of viral DNA into the nucleoprotein complex to form immature viral particles.

The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the Escherichia coli lacI operator/repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.

Animals↗

PubMed2000

Effect of body condition on reproductive efficiency of lactating dairy cows receiving a timed insemination.

Body condition may influence pregnancy rates to a timed insemination (Ovsynch/TAI) protocol and affect the economical performance of dairy farms. The objectives were to compare pregnancy rates using the Ovsynch/TAI protocol for the first service of lactating dairy cows with body condition scores < 2.5 (scale: 1 to 5, low BCS group) versus > or = 2.5 (control group) and to estimate the economic impact of the effect of body condition on reproductive performance. At 63 +/- 3 d post partum, cows were assigned to 2 experimental groups (low BCS = 81; control = 126), and were treated with GnRH at d 0 and with PGF2alpha 7 d later. At 48 h after PGF2alpha, cows received an injection of GnRH and were inseminated 16 h later. Pregnancy rates to the Ovsynch/TAI protocol were lower for the low BCS group than for the control group at 27 d (18.1 +/- 6.1% < 33.8 +/- 4.5%; P<0.02) and at 45 d (11.1 +/- 5.4% < 25.6 +/- 4.1%; P<0.02) after insemination. Economic analysis indicated that reducing the percentage of the herd in low body condition increases net revenues per cow per year. Body condition influenced pregnancy rates to the Ovsynch/TAI protocol.

Adipose Tissue↗

PubMed1999

The vaccinia virus 39-kDa protein forms a stable complex with the p4a/4a major core protein early in morphogenesis.

The vaccinia virus (VV) 39-kDa protein, the product of the A4L gene, is a highly antigenic protein of the viral core. Pulse-chase and immunoprecipitation experiments have shown that the 39-kDa protein interacts with p4a (encoded by the A10L gene), the precursor of the most abundant virion protein. This interaction is maintained with the processed 4a form that arises during virion maturation. The controlled disruption of mature viral particles showed that the 39-kDa and 4a proteins are tightly bound within the virion. Immunoelectron microscopy showed that both proteins first localize within the cytoplasm and later accumulate inside the viral factories, reaching these locations via a mechanism apparently unrelated to cellular membranes. Double labeling experiments showed a colocalization of both proteins in all virus-induced structures.

Animals↗

PubMed1999

Structural maturation of the transmissible gastroenteritis coronavirus.

During the life cycle of the transmissible gastroenteritis coronavirus (TGEV), two types of virus-related particles are detected in infected swine testis cells: large annular viruses and small dense viruses. We have studied the relationships between these two types of particles. Immunoelectron microscopy showed that they are closely related, since both large and small particles reacted equally with polyclonal and monoclonal antibodies specific for TGEV proteins. Monensin, a drug that selectively affects the Golgi complex, caused an accumulation of large annular viral particles in perinuclear elements of the endoplasmic reticulum-Golgi intermediate compartment. A partial reversion of the monensin blockade was obtained in both the absence and presence of cycloheximide, a drug that prevented the formation of new viral particles. After removal of monensin, the Golgi complex recovered its perinuclear location, and a decrease in the number of perinuclear large viral particles was observed. The release of small dense viral particles into secretory vesicles and the extracellular medium was also observed, as was a partial recovery of infectivity in culture supernatants. Small viral particles started to be seen between the third and the fourth Golgi cisternae of normally infected cells. All of these data strongly indicate that the large annular particles are the immature precursors of the small dense viruses, which are the infectious TGEV virions. The immature viral particles need to reach a particular location at the trans side of the Golgi stack to complete their morphological maturation.

Animals↗

PubMed1999

Visualization of viral assembly in the infected cell.

The study of the virus life cycle in infected cells is a methodological challenge due to the small size and diversity of the viral components. Recent developments on preservation of fine structure and molecular localization have provided a group of powerful methods with wide applications in cell biology and virology. Among the different electron microscopy (EM) techniques available to visualize viral assembly at the intracellular level, we will focus on conventional ultrathin sections, cryosections, and freeze-substitution. For obtaining molecular information associated to ultrastructure we have now a group of methods to detect viral proteins (immunogold labeling), as well as the viral genome, through the different techniques for detection of nucleic acids (the enzyme-gold approach, in situ hybridization, and elemental mapping). We will illustrate the applications of these methods with examples of viruses that exhibit different levels of structural complexity. These new approaches help to detect and identify viruses in clinical samples and to characterize the virus life cycle and the cellular components involved, to obtain data that could help for a therapeutic intervention, and to characterize virus-like particles that can be the basis of new and safe vaccines.

Animals↗

PubMed1998

Structure and intracellular assembly of the transmissible gastroenteritis coronavirus.

Coronaviruses have been described as pleomorphic, round particles with a helical nucleocapsid as the unique internal structure under the virion envelope. Our studies on the organization of the transmissible gastroenteritis coronavirus (TGEV) have shown that the structure of these viruses is more complex. Different electron microscopy techniques, including cryomicroscopy of vitrified viruses, revealed the existence of an internal core, most probably icosahedral, in TGEV virions. Disruption of these cores induced the release of elongated ribonucleoprotein complexes. Ultrastructural analysis of freeze-substituted TGEV-infected swine testis (ST) cells showed characteristic intracellular budding profiles as well as two types of virions. While large virions with an electron-dense internal periphery are seen at perinuclear regions, smaller viral particles exhibiting compact internal cores of poligonal contours are more abundant in areas closer to the plasma membrane of the cell. These data strongly suggest that maturation events following the budding process are responsible for the formation of the internal core shell, the new structural element that we have recently described in extracellular infectious TGEV virions.

Animals↗

PubMed1998

Use of plasma concentrations of 13,14-dihydro,15-keto-PGF2 alpha (PGFM) in the diagnosis of sub-clinical endometritis and its relationship to fertility in the postpartum dairy cow.

The objective of this study was to determine the value of using plasma concentrations of PGFM to diagnose subclinical endometritis in the dairy cow, and its relationship to subsequent fertility. A total of 274 cows between 24 to 29 d post partum was divided into 4 groups on the basis of clinical features of the uterus and ovary. Cows in Group 1 (n = 74) had a normal, involuting uterus and a CL on the ovary; cows in Group 2 (n = 51) had a normal, involuting uterus but no CL on the ovary; cows in Group 3 (n = 83) did not have a normal, involuting uterus but had a CL on the ovary; and cows in Group 4 (n = 66) did not have a normal, involuting uterus or a CL on the ovary. A blood sample was obtained from each cow on the day they were placed on the study, and plasma concentrations of PGFM and P4 were determined using RIA. Cows were artificially inseminated (AI) at the first observed estrus after Day 60 post partum, and pregnancy was determined by palpation of the uterus per rectum between 45 and 50 d postAI. Reproductive responses evaluated were conception rate to first service, days open, and percentage of cows pregnant by 90, 120, 150 and 180 d post partum. Data were analyzed using GLM procedures of SAS and a 2 x 2 factorial with contrast procedures. Polynomial regression analysis was used to determine the shape of the PGFM, P4 and fertility curves. There was no difference among mean PGFM concentrations of cows in each group. The rate of decline of plasma PGFM concentrations was lower in cows with an abnormal uterus and a CL on the ovary compared with those without a CL. A lower percentage of cows with abnormal uteri was pregnant by 90 d post partum compared with cows with normal uteri. From the results of this study, it was concluded that plasma PGFM concentrations between Days 24 to 29 post partum were not effective in identifying cows with subclinical endometritis.

Animals↗

PubMed1998

Reproductive performance of lactating dairy cows treated with gonadotrophin-releasing hormone (GnRH) and/or prostaglandin F2a (PGF2a) for synchronization of estrus and ovulation.

The objective of this study was to determine the reproductive performance of lactating dairy cows treated with GnRH and/or PGF2a for synchronization of estrus and ovulation. Between Days 43 and 57 post partum, a total of 374 dairy cows was divided into 4 groups. Cows in Group 1 (n = 62) were treated with 25 mg, i.m. PGF2a on Days 43 and 57; cows in Group 2 (n = 65) were not treated at this time; cows in Group 3 (n = 118) were treated with 100 ug, i.m. GnRH on Day 50, 25 mg, i.m. PGF2a on Day 57, 100 ug, i.m. GnRH on Day 59, and time-inseminated 16 h later; cows in Group 4 (n = 129) were treated with 25 mg, i.m. PGF2a once on Day 57. Cows in Groups 1 and 4 were inseminated at an induced estrus within 7 d after the last PGF2a treatment, and cows in Group 2 were inseminated at a noninduced estrus within a corresponding period of time. Conception rate, estrus detection rate and pregnancy rate were analyzed using logistic regression, and controlled for lactation number, body condition score and time of year. Days from calving to conception were analyzed using the GLM procedures of SAS, and the model included group, body condition score, lactation number, time of year, and their interactions. Cows in Group 3 had a significantly higher pregnancy rate than cows in Groups 1, 2 and 4. Orthogonal contrasts of mean days from calving to conception showed that cows in Group 3 had significantly (P < 0.01) less days from calving to conception than cows in Group 1 and Group 4. There was a significant effect of time of year on pregnancy rate and days from calving to conception, but there was no interaction between time of year and these reproductive characteristics. There was no effect of body condition score and lactation number on the reproductive characteristics evaluated. From the results of this study, it was concluded that better reproductive performance was observed in cows inseminated at a synchronized ovulation than in those inseminated at a synchronized estrous period.

Animals↗

PubMed1998

Optimization of phosphorus localization by EFTEM of nucleic acid containing structures.

Energy Filtered Transmission Electron Microscopy (EFTEM) has been used to study nucleic acids localization in unstained thin sections of virus-infected cells. For this purpose, phosphorus maps (P-maps) have been obtained by applying the N-windows Egerton model for background subtraction from data acquired by a non-dedicated TEM Jeol 1200EXII equipped with a post-column PEELS Gatan 666-9000 and a Gatan Image Filter (GIF-100). To prevent possible errors in the evaluation of elemental maps and thus incorrect nucleic acid localization, we have studied different regions of swine testis (ST) cells with similar local density containing either high concentration of nucleic acids (condensed chromatin and ribosomes) or a very low concentration (mitochondria). Special care was taken to optimize the sample preparation conditions to avoid as much as possible the traditional artifacts derived from this source. Selection of the best set of pre-edge images for background fitting was also considered in order to produce "true P-maps". A new software for interactive processing of images series has been applied to estimate this set. Multivariate Statistical Analysis was used as a filtering tool to separate the "useful information" present in the inelastic image series (characteristic signal) from the "non-useful information" (noise and acquisition artifacts). The reconstitution of the original image series preserving mainly the useful information allowed the computation of P-maps with improved signal-to-noise ratio (SNR). This methodology has been applied to study the RNA content of maturation intermediate coronavirus particles found inside infected cells.

Animals↗

PubMed1998

The fracture-flip technique reveals new structural features of the Escherichia coli cell wall.

With few exceptions, all bacteria possess a wall which protects them and controls their communication with the environment. In Gram-negative bacteria the cell wall exhibits a complex and unique multilayered organization. We have applied a modification of the freeze-fracture technique known as 'fracture-flip' to visualize the real surfaces of the different wall layers in a Gram-negative bacterium, Escherichia coli. In combination with treatments to weaken the interlayer connections, this technique has provided new insights into the structure of the bacterial wall. Large areas of an intermediate layer (most probably the peptidoglycan-containing matrix) have been visualized for the first time between the plasma membrane and the outer membrane of the wall. Extensive regions corresponding to the cytoplasmic face of the plasma membrane have also been obtained. These images provide new three-dimensional views of the bacterial cell wall and provide the structural framework for the analysis of the molecular relationships between the different cell wall components.

Cell Wall↗

PubMed1998

Expression of ORF A1 of infectious bursal disease virus results in the formation of virus-like particles.

A recombinant vaccinia virus inducibly expressing ORF A1 of infectious bursal disease virus (IBDV) has been constructed and characterized. Cells infected with this recombinant virus express the IBDV polyprotein, which is proteolytically processed to give mature VP2, VP3, and VP4 polypeptides. An electron microscopy study revealed that the cytoplasm of cells infected with the recombinant virus contains abundant IBDV-like particles (VLP). These VLP form close-packed paracrystalline arrays that are specifically recognized by anti-IBDV antibodies. The size and morphology of purified VLP were found to be akin to those of authentic IBDV particles.

Amino Acid Sequence↗

PubMed1998

Vaccinia virus 15-kilodalton (A14L) protein is essential for assembly and attachment of viral crescents to virosomes.

Early stages in vaccinia virus (VV) assembly involve the recruitment of cellular membranes from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) to virus factories (or virosomes). The key viral factors involved in this process are not yet known. We have previously identified and characterized two viral proteins, of 21 kDa (A17L gene) and 15 kDa (A14L gene), that associate with tubulovesicular elements related to the ERGIC and are localized in viral membranes at all stages of virion assembly. We showed that the 21-kDa protein is not responsible for the recruitment of membranes from the ERGIC to viral factories. However, it appears to be essential for the organization of viral membranes. In this investigation we have generated a VV recombinant, VVindA14L, in which the expression of the A14L gene is inducibly regulated by the Escherichia coli lacI operator-repressor system. Repression of 15-kDa protein synthesis has a dramatic effect on virus yields and severely impairs plaque formation. Compared to wild-type VV, reduced amounts of 15-kDa protein are produced in VVindA14L-infected cells in the presence of IPTG (isopropyl-beta-D-thiogalactoside), and this correlates with a small-plaque phenotype and reduced VVindA14L yields under these conditions. In the absence of the 15-kDa protein, early and late viral protein syntheses proceed normally; however, proteolytic cleavage of the major core precursors is inhibited. Electron microscopic examination of cells infected with VVindA14L under nonpermissive conditions reveals the presence of numerous membranous elements that look like unfinished or disassembled crescents interspersed between electron-dense masses. These abnormal membrane elements are usually well separated from the surfaces of the dense structures. These findings show that the 15-kDa protein is essential for VV morphogenesis and indicate that this polypeptide is necessary both for the correct assembly of viral crescents and for their stable attachment to the surfaces of viral factories.

HeLa Cells↗

PubMed1998

Two types of virus-related particles are found during transmissible gastroenteritis virus morphogenesis.

The intracellular assembly of the transmissible gastroenteritis coronavirus (TGEV) was studied in infected swine testis (ST) cells at different postinfection times by using ultrathin sections of conventionally embedded infected cells, freeze-substitution, and methods for detecting viral proteins and RNA at the electron microscopy level. This ultrastructural analysis was focused on the identification of the different viral components that assemble in infected cells, in particular the spherical, potentially icosahedral internal core, a new structural element of the extracellular infectious coronavirus recently characterized by our group. Typical budding profiles and two types of virion-related particles were detected in TGEV-infected cells. While large virions with an electron-dense internal periphery and a clear central area are abundant at perinuclear regions, smaller viral particles, with the characteristic morphology of extracellular virions (exhibiting compact internal cores with polygonal contours) accumulate inside secretory vesicles that reach the plasma membrane. The two types of virions coexist in the Golgi complex of infected ST cells. In nocodazole-treated infected cells, the two types of virions coexist in altered Golgi stacks, while the large secretory vesicles filled with virions found in normal infections are not detected in this case. Treatment of infected cells with the Golgi complex-disrupting agent brefeldin A induced the accumulation of large virions in the cisternae that form by fusion of different membranous compartments. These data, together with the distribution of both types of virions in different cellular compartments, strongly suggest that the large virions are the precursors of the small viral particles and that their transport through a functional Golgi complex is necessary for viral maturation.

Animals↗