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PubMed1982

Effect of different extenders on glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) release from frozen buffalo semen.

Sixty buffalo semen samples (motility greater than 60%) were frozen in 3 extenders, viz., Tris yolk glycerol (TY-G), Citric acid whey glycerol (CAW-G) and Egg yolk glucose sodium bicarbonate glycerol (EYGSB-G) for studying the release of GOT and GPT enzymes in the extracellular fluid during pre-freezing (after first extension) and post-freezing (15 minutes and 30 days after freezing). Release of GOT and GPT enzymes was less in TY-G than CAW-G and EYGSB-G extenders. Significant differences (P<0.01) in GOT and GPT release were observed between extenders and bulls at various stages of freezing of semen.

Journal Article↗

PubMed1985

Inclusion of bovine serum albumin in semen extenders to enhance maintenance of stallion sperm viability.

Semen from seven mature stallions was used to test the motility response of sperm cells when 3% bovine serum albumin (BSA) was added to seminal plasma and skim milk diluents. A total of 45 ejaculates was collected by artificial vagina and immediately evaluated for percent motile spermatozoa (PMS), rate of forward movement (RFM) and sperm cell concentration. Aliquots (four from each ejaculate) of raw semen containing 500x10(6) sperm cells were exposed to each of the following treatments: (1) seminal plasma (SP), (2) SP+BSA, (3) skim milk (SKM), (4) SKM+BSA; and incubated in 50-ml tubes at 37 C. The sperm cell characteristics, PMS and RFM, of each treatment suspension were reevaluated at 0, 0.5, 1, 2, 6, 12, 18 and 24 hr post-treatment. Inclusion of BSA and the type of extender, either seminal plasma or skim milk, significantly (P<0.05) affected the PMS and RFM of spermatozoa. Analysis of means within evaluation times showed that PMS maintenance was enhanced (P<0.05) when BSA was included in extenders at all incubation intervals except 24 hr. SKM+BSA maintained the highest (P<0.05) PMS for the first 2 hr with SP+BSA sustaining the highest (P<0.05) PMS from 12 to 24 hr. Skim milk alone sustained higher (P<0.05) PMS than the SP diluent for the first 6 hr of incubation, whereas SP maintained a higher (P<0.05) PMS than SKM from 18 to 24 hr. The RFM of spermatozoa was greatest (P<0.05) for the first 6 hr of incubation when exposed to SKM+BSA. Seminal plasma + BSA sustained a higher (P<0.05) RFM for the first 6 hr of incubation than SP alone, but not higher than SKM at this interval. Skim milk sustained a higher (P<0.05) RFM of spermatozoa for the first 6 hr of incubation than SP. These data support the hypothesis that BSA protects spermatozoa from the harmful effects of lipid peroxidation. Including this substance in semen extenders may prolong maintenance of sperm motility.

Journal Article↗

PubMed1988

Fluorometric evaluation of cryopreserved bovine spermatozoa extended in egg yolk and milk.

A comparison of fluorogenically quantifiable parameters of cryopreserved, bovine spermatozoa that had been processed in homogenized milk and egg yolk citrate-based extenders was made using flow cytometry. Semen from four bulls was processed in egg yolk-citrate or homogenized milk extenders, packaged in straws and frozen at -196 degrees C. Samples were thawed at 37 degrees C, subdivided into three portions and stained after 0, 1.5 and 3 h of incubation at 37 degrees C. Spermatozoa were stained using a combination of carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) and analyzed by dual parameter flow cytometry. The sperm cells were quantified fluorometrically at each time interval for both green and red fluorescence. The proportion of spermatozoa retaining the fluorescent CFDA derivative was larger at each time interval for samples in egg yolk citrate than those in milk. Differences in the retention of spermatozoal viability were detected between identical samples of bovine spermatozoa extended in milk or egg yolk based media.

Journal Article↗

PubMed1990

Freezability, enzyme leakage and fertility of buffalo spermatozoa in relation to the quality of semen ejaculates and extenders.

Forty-eight semen ejaculates from four Surti buffalo bulls were studied under split sample technique to establish the effects of initial semen quality and tris fructose yolk glycerol (TFYF), egg yolk citrate glycerol (EYCG) and lactose yolk glycerol (LYG) extenders on the freezability, fertility (based on 3412 AI) and extracellular release of spermatozoal enzymes pre and postfreezing. The overall mean activity of GOT, GPT, AKP, ACP and LDH enzymes in the postthaw seminal plasma increased significantly (P<0.01) above prefreeze levels, whereas sperm motility decreased. Freezability and the release of these enzymes were not influenced by the types of extenders used except for AKP release, which was significantly (P<0.01) lower in TFYG diluent. The pre and postfreeze sperm motility was significantly higher (P<0.01) and the leakage of GOT, AKP and ACP was lower in 36 semen samples with an initial motility above 70% than in the 12 samples in which initial motility was between 60 and 70%. The effects of interactions between motility groups, diluents and freezing periods were statistically nonsignificant for both freezability and leakage of all five enzymes. Fertility rate of frozen semen produced in TFYG diluent was significantly (P<0.05) higher (42.69%) than in the other diluents and was followed by that of EYCG (39.78%) and LYG (37.50%) with an overall mean of 40%. Sperm post-thaw motility had significantly (P<0.01) negative correlations with the release of enzymes: GOT (-0.948); GPT (-0.859); AKP (-0.673); ACP (-0.951) and LDH (-0.764). Fertility rates showed high negative correlations with the release of all five enzymes. Freezability was positively correlated with fertility (+0.405). Significant positive correlations were also observed for the release of GOT with that of GPT (+0.944); AKP (+0.574); ACP (+0.911) and LDH (+0.839): GPT with ACP (+0.795) and LDH (+0.870): and ACP with AKP (+0.725) and LDH (+0.577). These findings stressed the use of simple estimates of GOT and AKP leakage as markers for the assessment of freezability and fertility, and also the importance of initial good quality semen and the suitablity of extenders (TFYG) in the production of frozen buffalo bull semen for better fertility rates.

Journal Article↗

PubMed1990

Effect of storage temperature, cooling rates and two different semen extenders on canine spermatozoal motility.

Ejaculates were collected form three mixed-breed male dogs daily for 3 d. The semen was diluted in either a nonfat dried milk solid-glucose (NFDMS-G) or egg yolk citrate (EYC) extender at a concentration of 25 x 10(6) sperm/ml. The diluted samples were exposed to three different storage temperatures (35, 22 and 4 degrees C). Three cooling rates (-1.0, -0.3 and -0.1 degrees C/min) were also investigated at the lowest storage temperature (4 degrees C). The semen was evaluated for total motility, progressive motility and velocity at 0, 6, 12, 24, 48, 72, 96 and 120 h after collection by two independent observers. Interactions between extenders, temperatures and time after collection were found for each of the variables. Nonfat dried milk solid-glucose diluent was superior to EYC (P<0.05) in preservating sperm motility parameters that were evaluated for most of the observations. The evaluated sperm motility parameters were also significantly superior (P<0.05) in semen stored at 4 degrees C than at 35 or 22 degrees C for most of the observations. The progressive motility and velocity of sperm in semen cooled at 4 degrees C in NFDMS-G were higher (P<0.05) at the fast and medium cooling rates (-1.0 and -0.3 degrees C) than at the slow cooling rate (-0.1 degrees C/min) at 24 and 72 h, and at 48 h, respectively. In conclusion, the present study suggests that canine spermatozoal motility is well preserved when a NFDMS-glucose extender is added to the semen and the semen is cooled at a medium or fast rate to a storage temperature of 4 degrees C. Additional studies are needed to evaluate the fertility of semen stored in this manner.

Journal Article↗

PubMed1989

Polymorphism of three HLA-DR7 bearing major histocompatibility complex extended haplotypes.

We have studied the complexity of HLA class II region in DR7 bearing extended haplotypes by restriction fragment length polymorphism (RFLP). Genomic DNA from homozygous cell lines and from unrelated individuals was digested with a number of restriction endonucleases and probed with DR alpha, DR beta, DQ alpha and DQ beta cDNA probes. We detected RFLPs that distinguished subspecificities of DRA, DRB1, DQA1 and DQB1 chain genes. On the basis of polymorphism in these genes, three distinct types of DR7 bearing extended haplotypes could be identified: (1) B44 or Bw47 or B14, DR7a (DRA1, B1.1), DRw53a (DRA1,B4), DQw2 (DQA1.1, B1.1); (2) B13 or B40, DR7b (DRA1, B1.2), DRw53a (DRA1, B4), DQw2 (DQA1.1, B1.1); and (3) Bw57, DR7c (DRA2, B1.2), DRw53b (DRA2, B4), DQw9 (DQA1.2, B1.2). Available evidence indicates that independent examples belonging to a haplotype were similar in RFLP patterns, suggesting that most examples of an extended haplotype belonging to a subtype are similar. The results in the present study have important implications for immune function and disease susceptibility.

Blotting, Southern↗

PubMed1990

Comparison of fortified calf serum, serum substitutes and fetal calf serum with or without extenders for propagation of cell cultures for virus plaque assays.

Two studies comparing sewage-isolated and laboratory stock viruses were conducted to determine if alternative forms of serum or serum extenders could be used in place of fetal bovine serum without a significant loss of viral titer. In the first study, BGM cells were grown in standard MEM-L15 medium which was supplemented with Nuserum, Sigma serum replacement (CPSR-1), HyClone defined iron supplemented calf bovine serum, fetal bovine serum (FBS) or FBS supplemented with either SerXtend or Mito serum extenders. Comparison of virus titers showed that CPSR-1 gave the best overall results and was comparable to FBS. Of the serum extenders, only SerXtend improved virus recovery from sewage samples. In the second study, all sera were tested with and without SerXtend. In these experiments, SerXtend enhanced virus sensitivity of the BGM cell line grown in the HyClone serum but reduced the sensitivity of those cultured in Sigma serum. In both series, the growth of BGM cells was monitored for 12 weeks and all test products were shown to support long-term cell growth.

Analysis of Variance↗

PubMed1990

N-terminally extended substance P is released together with substance P from rat spinal cord.

The release of different forms of substance P-like immunoreactivity (SP-LI) from superfused slices of rat spinal cord was studied. The released SP-LI was characterized by reverse-phase high-performance liquid chromatography and radioimmunoassay with two antisera directed to the C- and N-terminal parts of SP, respectively. The SP-LI detected in the superfusates with the C-terminally directed antiserum was found to consist of (undeca) SP, SP-sulfoxide and a late eluting component which was not detectable with the N-terminally directed antiserum. This component was also found in neutral extracts of the spinal cord. Upon trypsin digestion, it produced SP-LI detectable with both C- and N-terminally directed antiserum which also coeluted with SP. From these results we conclude that this form of SP-LI most likely corresponds to an N-terminally extended form of SP. An increase of the potassium concentration in the superfusion fluid from 5 to 50 mM evoked an increased overflow of both SP and the N-terminally extended SP. The present results indicate that N-terminally extended SP is released by a calcium-dependent mechanism together with SP from terminals in the spinal cord in response to potassium stimulation.

Animals↗

PubMed1992

Readthrough of the Bacillus subtilis stop codon produces an extended enzyme displaying a higher polymerase activity.

It has been generally accepted that the structural sacB gene of Bacillus subtilis levansucrase encodes a 50,000 Da extracellular protein. However, examination of the DNA sequence of the sacB flanking regions shows a putative open reading frame coding for a 20 amino acid peptide downstream immediately following the terminal TAA stop codon. By site-directed mutagenesis we have changed this stop codon to a glutamine codon. This stop codon readthrough leads to the synthesis and secretion by B. subtilis of a levansucrase possessing an extended polypeptide chain. The extended levansucrase has a molecular weight of 53,000 with a new carboxyl-terminus, rich in basic and hydrophobic amino acids and possessing one cysteine residue. This enzyme synthesizes fructosyl polymer levan of higher molecular weight than the shorter levansucrase. The increase in molecular weight was achieved by increasing the number of branches. These results suggest that the C-terminal part of the enzyme plays a specific role in the degree of branching of the synthesized polymer. Moreover, the extended enzyme is able to form an active dimer from two polypeptide chains linked by an S-S bridge.

Amino Acid Sequence↗

PubMed1995

Role of prolonged surveillance in the eradication of nosocomial scabies in an extended care Veterans Affairs medical center.

Although general guidelines for control of institutional outbreaks of scabies have been published, little information is available on the long-term efficacy of these measures in extended care facilities. An epidemic of scabies occurred in a comprehensive care Veterans Affairs facility as a result of an unrecognized case of crusted scabies, with a total of 112 persons affected during a 12-month period. The initial outbreak occurred in the acute care units, with highest attack rates among roommates of the index patient (11/14, 78%) and nursing staff (27/55, 49%). Despite sustained infection control measures, secondary outbreaks continued to occur in the extended care units. Factors contributing to the persistence of the epidemic were transfer of patients with unrecognized infestation within the facility, prolonged latency period and atypical manifestations in elderly patients, and failure of scabicide treatment. In addition, a role may be played by carriage of scabies mites by infested staff members before they have symptoms. Control of the epidemic was only achieved with the following: increased awareness and better scabies recognition, restriction of staff rotation in the facility, and improved communication among primary providers and infection control personnel. Prolonged surveillance may be required for eradication of nosocomial scabies in extended care settings.

Aged↗

PubMed1988

Two variants of DRw52, DR3, and DQw2 specificities distinguish two different DR3-bearing extended haplotypes.

HLA-DR beta and DQ beta MHC antigens present on B-lymphoblastoid cell lines homozygous for either [HLA-B8,DR3,SC01] or [HLA-B18,DR3,F1C30] were studied by two-dimensional gel electrophoresis. Comparison of neuraminidase-treated DR proteins from these cells showed that DR3-bearing cells express two DR beta genes. However, one DR beta chain (beta a) is nonpolymorphic, whereas the other beta chain (beta b) is polymorphic. Two variants of DR beta (DR3 or DRw52) and two of DQ beta (DQw2) were found, with all the examples of each extended haplotype carrying only one of these sets of variants. The variants thus absolutely distinguished the two DR3-bearing extended haplotypes. These results support the hypothesis of extended haplotypes at the protein level, and demonstrate the fixity of alleles on them in the HLA-B-D/DR region.

Antigen-Antibody Complex↗

PubMed1984

Extended field irradiation in the treatment of patients with cervical carcinoma involving biopsy proven para-aortic nodes.

Between November 1974 and November 1979, 15 patients with cervical carcinoma were treated with extended field irradiation for biopsy proven para-aortic lymph node (PALN) metastases. Treatment consisted of pelvic and para-aortic irradiation at a daily dose of 180 to 200 rad per day, delivering 4000 to 6000 rad to the pelvis and 4000 to 5000 rad to the para-aortic nodes. One or two intracavitary insertions each delivered an additional 2000 to 3500 rad to point A. The three year actual disease free survival for the 12 patients with Stage I and II disease was 50%. All six survivors remain alive without evidence of disease for 41 to 93 months, with a mean and median follow-up of 65 months. All patients dying of disease did so within 26 months, all but one dying within one year. All patients with Stage III and IV are dead of disease. Pelvic disease was controlled in 11 of 12 patients with Stage I or II disease, and in one of the three patients with Stage III and IV disease. There was no clinical indication of failure in the PALN in any patient. Nine patients failed with disseminated disease. Three of 15 patients (20%) suffered serious treatment-related complications. Two of these were attributed to the pelvic irradiation, with one patient requiring a colostomy. Thus, complications resulting from the extended field irradiation were seen in only one patient (6.7%). There was no treatment related mortality. Extended field irradiation can lead to a 50% survival in patients with Stage I and II cervical carcinoma and PALN metastases, a survival comparable to that reported in patients with involved pelvic nodes.

Adenocarcinoma↗

PubMed1986

Complications of extended-field therapy for cervical carcinoma without prior surgery.

This study is designed to analyze the complications of extended-field radiotherapy for carcinoma of the uterine cervix uncomplicated by recent prior surgery. Forty-two patients with carcinoma of the uterine cervix and lymph node metastases established by unequivocally positive bipedal lymphangiograms were treated with extended-field radiotherapy. External beam radiation to extended pelvic portals was limited to 4500 cGy using the linear accelerator and approximately 6000 mg-hr brachytherapy. Nodal boosts up to 500 cGy were generally limited to fields measuring less than 50 cm2. Higher doses were administered in 12 patients because of poor tumor regression. Eleven of these 12 patients experienced severe complications, and only three achieved control of their tumor. The type of treatment complication appeared to be directly related to specific modifications of the initial treatment plan. Treatment failures occurred within and outside of treatment portals with equal frequency.

Carcinoma↗

PubMed1995

Dose distribution considerations of medium energy electron beams at extended source-to-surface distance.

PURPOSE: To determine the effects of extended source-to-surface distance (SSD) on dose distributions for a range of medium energy electron beams and cone sizes. METHODS AND MATERIALS: The depth-dose curves and isodose distributions of 6 MeV, 10 MeV, and 14 MeV electron beams from a dual photon and multielectron energies linear accelerator were studied. To examine the influence of cone size, the smallest and the largest cone sizes available were used. Measurements were carried out in a water phantom with the water surface set at three different SSDs from 101 to 116 cm. RESULTS: In the region between the phantom surface and the depth of maximum dose, the depth-dose decreases as the SSD increases for all electron beam energies. The effects of extended SSD in the region beyond the depth of maximum dose are unobservable and, hence, considered minimal. Extended SSD effects are apparent for higher electron beam energy with small cone size causing the depth of maximum dose and the rapid dose fall-off region to shift deeper into the phantom. However, the change in the depth-dose curve is small. On the other hand, the rapid dose fall-off region is essentially unaltered when the large cone is used. The penumbra enlarges and electron beam flatness deteriorates with increasing SSD.

Electrons↗

PubMed1994

Dosimetric effects of abutting extended source to surface distance electron fields with photon fields in the treatment of head and neck cancers.

PURPOSE: In the management of head and neck cancer, it is often necessary to junction photon and electron fields. When patients are treated supine, electron cones or applicators may have to be positioned at source to surface distance (SSD) greater than the standard 100 cm because of the patient's shoulders. We have studied the dosimetric effects of abutting 6 MV photon fields with 9 MeV electron fields at extended SSDs to assess changes in the 90% isodose width, dose uniformity in the target, and the extent of hot and cold spots in the junction region. METHODS AND MATERIALS: Four independent film studies were conducted, in a polystyrene phantom, for evaluation of the dose distribution in the junction region. Measured distributions were also compared with computer generated distributions using a treatment planning computer system. RESULTS: At the junction line between photon and extended SSD electron beams, hot and cold spots were observed. A 20% hot spot occurred on the photon side because of the electron scatter from the adjoining field. The width of this hot spot increased in dimension but not in magnitude as electron SSDs were increased. The cold spot occurred on the electron side due to the decrease in 90% isodose width at extended electron SSDs. This cold spot was minimal (less than 10%) at shorter electron SSDs, but increased as electron SSDs approached 120 cm. Computer dosimetry underestimated these results because of limitations of the electron beam treatment planning algorithm. CONCLUSION: These hot and cold spots may be clinically acceptable compared to the loss of treatment accuracy and the added possibility of more extensive hot or cold spots if the patient were to be removed from the original supine position and repositioned on his/her side.

Electrons↗

PubMed1992

Incidence of two virulence factors (aerobactin and mucoid phenotype) among 190 clinical isolates of Klebsiella pneumoniae producing extended-spectrum beta-lactamase.

Because outbreaks of multiple-resistant Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamases were recently observed in French hospitals, the presence of virulence factors was examined for (i) phenotype by bioassay for aerobactin production and by culture for the mucoid phenotype, and (ii) genotype using intragenic probes of respectively 2-kb BglII and 235-bp BamHI-BglII fragments and dot-blotting among 190 unreplicated K. pneumoniae clinical isolates issued from 25 French hospitals and producing different types of extended-spectrum beta-lactamases (TEM-related enzymes: TEM-3, TEM-4, CAZ-1, CAZ-2, TEM-8, or SHV-related enzymes: SHV-2, SHV-3, SHV-4). Only 3.7% and 7% of K. pneumoniae isolates produced aerobactin and mucoid phenotypes respectively, unrelated to type of beta-lactamase. Only 2% had both factors. No discordance was reported according to the detection method tested. The low prevalence of such virulence factors seems to indicate they were not involved in dissemination of nosocomial K. pneumoniae isolates producing an extended-spectrum beta-lactamase.

Cross Infection↗

PubMed1996

Cloning and characterization of extended hammerheads from a diverse set of caudate amphibians.

Satellite 2 (sat2) elements from six species representing four caudate amphibian families were cloned and analyzed. Despite differences in the abundance of this element, the tandemly repeated genomic organization found for sat2 in earlier investigations was conserved in the newly investigated species. The ability of sat2 transcripts to catalyze their own site-specific self-cleavage using an extended hammerhead (HH) motif had also been conserved. This motif is similar to the HH found in a number of infectious plant RNAs, but has an abbreviated stem III region and an internally looped stem I extension that distinguish it from the prototypical HH. The extended HH analyzed in this survey can be further organized into two groups on the basis of shared sequences and structural details in the peripheral stem-loop structures. HH derived from the family Salamandridae constitute one group, whereas HH from all other salamanders that have been investigated belong to the other group. Although many of the natural variants did not cleave efficiently in vitro, examples of relatively active variants for each design were found. This survey has therefore identified two designs that are suitable starting models for the structural and functional analysis of the extended HH.

Amphibians↗

PubMedDate not supplied

The expression of the EF1 alpha genes of Drosophila is not associated with the extended longevity phenotype in a selected long-lived strain.

A quantitative dot blot analysis was performed to determine whether the expression of the EF-1 alpha genes of Drosophila melanogaster are associated with the extended longevity phenotype characteristic of our genetically selected long-lived strain. These data were compared to that obtained from two normal-lived strains and from two iso-chromosomal strains with an intermediate life span. The relative mRNA levels of both EF-1 alpha genes (EF-1 alpha F1 and EF-1 alpha F2) for all five strains were measured through the larval, pupal, and early adult stages, and statistically analyzed. Our findings from these studies indicate that the F2 mRNA tracks with the extended longevity; however, the F1 mRNA is the major component and, thus, the relative total expression of these genes at the mRNA level is approximately equivalent for all five strains. These conclusions suggest that the expression of the EF-1 alpha genes is not associated with the extended longevity phenotype.

Animals↗