A PCR detection method for testing Mycoplasma contamination of veterinary vaccines and biological products.

A rapid test method was developed for detecting mycoplasma contamination in veterinary biological products. The method reduces testing time by 2 weeks and shows comparable sensitivity to the current agar-based detection model. The primary goals for the development of the test were to reduce the testing time, incorporate a method that was easily adaptable across the veterinary biologics industry and reduce the subjective interpretation of results. We found that biological enrichment is necessary to maintain sensitivity of the detection method when compared to the standard culture-based test and that periodic sampling of enrichment cultures is essential to detect a wide variety of mycoplasma species that may be present as contaminants. The PCR detection method is comparable to the agar-based model and can reduce the overall testing time by up to 14 days.

DNA sequence encoding the amino-terminal region of the human c-src protein: implications of sequence divergence among src-type kinase oncogenes.

We sequenced the 5'-coding region of the human c-src gene, exons 2 through 5, corresponding to one-third of the human c-src protein consisting of 536 amino acids. Sequence analysis of the src type of protein kinases revealed that the amino-terminal region encoded by exon 2 contains sequences specific for the src proteins and raised the possibility that this region is involved in the recognition of a src-specific substrate(s) or receptor(s).

Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src.

The nucleotide sequence of the 3' two-thirds of a highly conserved, molecularly cloned human cellular src gene (c-src) has been determined. This region of the c-src gene encodes the tyrosine kinase domain of the cellular src protein (pp60c-src) and corresponds to exons 6 through 12 of the chicken c-src gene, as well as nucleotides 545 to 1542 of the Rous sarcoma virus src gene (v-src). The human c-src sequence is very strongly conserved with respect to both the chicken c-src and the Rous sarcoma virus v-src genes, with nearly 90% nucleotide homology observed in this region. Amino acid sequence conservation in this region is even greater; 98% of the amino acids are conserved between human and chicken c-src. Furthermore, the exon sizes and the locations of the exon-intron boundaries are identical in the human and chicken c-src genes. However, sequences within the introns have not been conserved, and the introns within the human c-src gene are significantly larger than the corresponding introns within the chicken c-src gene. The strong amino acid conservation between the carboxy-terminal two-thirds of pp60c-src of species as divergent as humans and chickens suggests that this portion of the pp60c-src protein specifies one or more functional domains that are of great importance to some aspect of normal cellular growth or differentiation.

Cloning and transcriptional analysis of a variant surface glycoprotein gene expression site in Trypanosoma brucei.

The variant surface glycoprotein (VSG) gene expression site in Trypanosoma brucei variant 117a has been mapped to a point about 40 kb upstream from the VSG gene. Sequences upstream from the previously identified [Cully, D.F. et al. (1985) Cell 42, 173-182] expression site associated gene (ESAG-I) have been cloned and a stable 1.3 kb transcript has been localized immediately 5' to ESAG-I. This transcript is in the same orientation and approximately as abundant as the ESAG-I message. A highly conserved region, of which at least 15 copies are present in the genome, has been identified further upstream. A stable transcript corresponding to this region was not detected in variant 117a, but a 1.7 kb transcript was detected in variant 221a. In isolated nuclei, representative sequences from the 117a, expression site were transcribed unidirectionally at similar rates, and transcription was insensitive to alpha-amanitin.

Identification of proteins encoded by variant surface glycoprotein expression site-associated genes in Trypanosoma brucei.

The variant surface glycoprotein (VSG) genes of Trypanosoma brucei may be transcribed from several distinct telomeric expression sites (ESs). The mechanism responsible for regulating potential expression sites is unknown. Two members of a pleomorphic family of expression site associated genes (ESAGs) have been cloned and sequenced. By examination of the DNA sequences we inferred that ESAGs encode amphiphilic glycoproteins. Fragments of two ESAGs were inserted into the Escherichia coli expression vectors pATH and pEX. Antisera to the resulting anthranilate synthetase ESAG protein (ESAGP) fusion protein immune precipitated a 46 kDa glycoprotein from detergent extracts of T. brucei. In the presence of tunicamycin, the size of the immune-precipitated protein was reduced to 36 kDa, corresponding to the molecular weight predicted by the ESAG sequence. The 36 kDa and 46 kDa proteins were absent from procyclic culture forms of T. brucei.

Castrating operations associated with metastatic lesions of the breast.

PIP: To determine whether subsequent pelvic difficulty justified hysterectomy in cases of surgical castration performed as treatment of breast cancer, 432 cases so operated on at the Mayo Clinic for the 13 years 1955-1967 were reviewed. Oophorectomy alone was done in 353 patients, oophorectomy plus hysterectomy in 79. The age range in the oophorectomy group was 18-59 years, in the hysterectomy group 28-63 years. In each, 55% of the patients were less than 50-years-old. Of 367 patients in whom the procedure was therapeutic, 88 were found to have metastatic carcinoma in 1 or both ovaries. Inspection at the operating table had failed to reveal these metastases. Of the 79 uteri removed, 74 had demonstrable pathologic process. In 7 cases lesions were malignant. Postoperative vaginal bleeding occurred later in 14 of the 353 cases in which the uterus had been left in place. Morbidity rates, as determined by fever on 2 postoperative days, were 44% in the hysterectomy group, 26% in the oophorectomy group. Cause of the fever was not determined in half of these cases. Each group included a single case of afebrile thrombophlebitis. Hospital stay averaged 8 days for oophorectomy, 10 days for hysterectomy patients. Castration of the premenopausal patient with metastatic breast cancer offers a chance of remission. About a third of such patients are benefitted and survival may be increased 2 years. Surgical castration is preferred over irradiation because it gives more rapid hormone withdrawal. All ovarian tissue must be removed, especially when adhesions or endometriosis is present. Hysterectomy at the time of oophorectomy is considered justified except in poor-risk patients.^ieng

Genome plasticity in Neisseria gonorrhoeae.

The pathogenic Neisseria have exploited the processes of horizontal DNA transfer and genetic recombination as mechanisms for the generation of extensive protein variation and modulation of gene expression. Localized recombinations have been well documented in members of multigene families as have alterations in short repetitive sequences. Here we report an analysis of the chromosomal structure of a defined lineage of Neisseria gonorrhoeae strain MSl1 pilin variants. This study reveals the occurrence of large rearrangements, including the amplification of a 26 kb region and an inversion involving more than a third of the chromosome. Additionally, a restriction site polymorphism that correlates with pilin expression has been observed. These findings highlight the flexibility of the gonococcal genome.

Oncological outcomes of operative treatment of subcutaneous soft-tissue sarcomas of the extremities.

We reviewed the cases of sixty-two patients who had had a subcutaneous sarcoma to determine the effect of tumor and treatment-related variables on the rates of survival and local recurrence. Fifty-nine (95 per cent) of the patients had had an operation at another hospital before being referred to us. Twenty-nine (47 per cent) of the sixty-two tumors were high-grade, forty-two (68 per cent) were small (five centimeters or less), and thirty (48 per cent) were malignant fibrous histiocytomas. We followed a treatment strategy that consisted of repeat excision with the goal of obtaining wide margins. Excluding thirteen patients who had had a palpable local recurrence at the time of presentation, twenty (49 per cent) of forty-one patients who had had a marginal excision at another hospital had microscopic residual tumor on repeat excision. At a median of fifty-six months after the repeat excision, fifty (81 per cent) of the sixty-two patients had been continuously disease-free, one had no evidence of disease, eight had died of the disease, and three had died of other causes. The five-year rate of disease-free survival was 85 per cent (fifty-three of sixty-two patients). There were three local recurrences, all in patients who had had a marginal resection. No recurrences were noted in patients who had had a wide local excision of the tumor or of the previous operative field. Multivariate analysis revealed that a large tumor (greater than five centimeters), a marginal excision, and adjuvant radiation therapy were associated with a worse prognosis. Excellent rates of survival for patients who have a subcutaneous sarcoma, including those who have a large or high-grade tumor and those who have residual tumor following a previous operation, can be obtained with carefully planned operative treatment alone. We recommend operative excision or repeat excision with wide margins because of the high prevalence of residual tumor. Size is the most important tumor-related factor, and the operative margin is the most important treatment-related factor. The additional value of adjuvant radiation therapy remains unproved.

Aneurysmal bone cyst of the extremities. Factors related to local recurrence after curettage with a high-speed burr.

BACKGROUND: Aneurysmal bone cyst is a benign, locally destructive lesion of bone. The rates of local recurrence after curettage have varied widely. Therefore, we performed a retrospective study of patients who had had an aneurysmal bone cyst in order to identify the rate of local recurrence and the prognostic factors related to local recurrence after use of contemporary methods of curettage with a high-speed burr. METHODS: We reviewed the cases of forty patients who had been managed by the same surgeon for an aneurysmal bone cyst, as diagnosed on the basis of the latest pathological review, between January 1, 1976, and December 31, 1993. The patients were evaluated with regard to age, gender, the duration and type of symptoms, the presence or absence of pathological fracture, the status of the growth plate, the bone and part of the bone that were involved, the type of operative procedure, the outcome, the radiographic stage, the findings on magnetic resonance imaging and computerized tomography (when it became available) and on bone scintigraphy, and histological parameters. The median duration of follow-up was eighty-seven months (range, fifteen to 267 months). According to the criteria of Enneking, no patient had a stage-1 lesion (one with a surrounding rim of cortical bone), twenty-four had a stage-2 lesion (one with a clearly defined border but no cortical bone), and sixteen had a stage-3 lesion (one with no clearly defined border). RESULTS: Of the forty patients, thirty-four had curettage with use of a high-speed burr. Of these thirty-four, twenty-two had filling of the defect with a cancellous autogenous graft; four, with a cancellous allograft; and three, with polymethylmethacrylate. In five patients, no material was put into the defect. The remaining six patients had resection through the margin of the lesion. Four (12 percent) of the thirty-four patients who had curettage had a local recurrence. No patient who had an excision through the margin of the lesion had a local recurrence. All local recurrences were in skeletally immature girls who were three, four, ten, and eleven years old. Univariate analysis with use of the chi-square, Fisher exact, and Wilcoxon log-rank tests showed that local recurrence was associated only with a young age (p = 0.0036) and open growth plates (p = 0.039). All local recurrences occurred within two years postoperatively, at two, seven, nine, and twenty-four months, and all were treated successfully with a second operation. CONCLUSIONS: Rates of local control of almost 90 percent can be achieved with thorough curettage with use of a mechanical burr and without use of liquid nitrogen, phenol, or other adjuvants in patients who have an aneurysmal bone cyst of an extremity. A young age and open growth plates are associated with an increased risk of local recurrence.

Oral intake policies on labor and delivery: a national survey.

STUDY OBJECTIVE: To examine current policies on oral intake during labor among hospitals throughout the United States. DESIGN AND SETTING: Anonymous questionnaire survey distributed to the directors of anesthesia and obstetrics departments of 740 hospitals. Completed surveys were then grouped by number of deliveries performed each year. MEASUREMENTS AND MAIN RESULTS: A total of 2,265 surveys were distributed. Of that number, 902 (33% response rate) surveys, representing 740 U.S. hospitals, were returned. Of the surveys returned, 419 surveys were received from obstetricians and 401 surveys were received from anesthesiologists. Oral intake during labor is limited primarily to clear liquids, although hospitals with fewer deliveries allow significantly more oral intake during latent phase than do hospitals with larger services. Allowing nonclear liquids or solid foods is uncommon in either phase of labor, regardless of hospital size. CONCLUSIONS: The results give an indication of oral intake policies used by labor and delivery units in the United States, and they may be helpful for obstetric services that are in the process of developing their own policies.

Periosteal chondroma at birth.

Periosteal chondroma is a slow-growing, cartilaginous, surface tumor that usually occurs in the second and third decades of life. The youngest reported age at diagnosis is 5 years. Marginal excision is the treatment of choice. We report a case of a periosteal chondroma noted at birth and treated conservatively. This report expands the age range of periosteal chondroma to include neonates and suggests a role for observation in its management.

Training anesthesia personnel to perform bleeding times.

Template bleeding time results generated by residents in anesthesiology correlate well with those obtained by trained laboratory personnel (p less than 0.002). Anesthesia personnel can accurately perform bleeding time tests when laboratory support is not available. The results of these tests can be used to guide clinical decisions.

Ovarian blood flow in conscious and anaesthetized pregnant rabbits near term and the influence of arterial blood gas tensions.

Total ovarian, luteal and stromal blood flows were measured with radioactive microspheres (25 mum diameter) in 7 rabbits anaesthetized with sodium pentobarbitone and 23 conscious rabbits at Day 28 of gestation. Despite major differences in cardiac output, arterial PO2, PCO2, pH and base status, ovarian tissue blood flows were similar in both groups. In the conscious rabbits total ovarian blood flow was negatively related to arterial pressure, and luteal blood flow was negatively related to pressure and arterial pH. In the anaesthetized rabbits ovarian stromal blood flow was positively related to arterial PO2.

Human uterine artery responses to lidocaine.

Uterine arteries from pregnant and nonpregnant patients were studied to define their responses to lidocaine. Although local anesthetics usually are considered vasodilators, these data indicate that the human uterine artery constricts when exposed to 1,000 mug of lidocaine in vitro. Furthermore, those arteries from pregnant patients exhibited significantly greater responses than did those from nonpregnant patients.

Response of arterial segments from gravid human uterus to multiple concentrations of lignocaine.

Nine uterine artery segments were obtained during Caesarean hysterotomy. The amount of isometric tension developed by each segment when exposed to 3, 6, 10, 20, 50, 100, 1000 and 2000 microng/ml of lignocaine hydrochloride (Xylocaine) was measured. All nine segments contracted in response to the 1000- and 2000-microng/ml concentrations. Eight of nine, six of nine, and three of nine responded to the 100-, 50- and 20-microng/ml concentrations, respectively; one segment contracted in response to all concentrations. A dose-response curve indicated that maximum responses were obtained at the 1000-microng/ml concentration (0.1%). Although local anaesthetics are usually considered vasodilators, this study indicates that lignocaine causes the uterine artery to constrict in concentrations in excess of 20 microng/ml. Such concentrations might occur after paracervical block or accidental intravascular injection, but would not be expected in association with extradural analgesia.

Evidence for lateral transfer and recombination in OspC variation in Lyme disease Borrelia.

The ospC gene was amplified by the polymerase chain reaction from each of 76 Lyme disease Borrelia strains. Restriction fragment length polymorphism (RFLP) analysis demonstrated 33 distinct RFLP types; two additional RFLP types were identified from published ospC sequences. For each RFLP type, at least one ospC gene was sequenced and the degree of sequence relatedness examined by construction of an ospC gene tree. The genes were extremely diverse, with sequence identity ranging from 74.4% to 99.0%; the majority of changes are localized within the central portion of the molecule. A comparison of ospC sequences suggests that recombination occurs frequently between ospC alleles; this genetic exchange is proposed to be mediated by lateral transfer of ospC sequences. Evidence indicates that recombination occurs between ospC genes from the same Borrelia species (i.e. B. afzelii and B. garinii) as well as between different Borrelia species (i.e. B. afzelii and B. garinii, B. burgdorferi and genogroup DN127).

Anesthetic solubility coefficients for maternal and fetal blood.

Solubility coefficients for seven inhalation anesthetic agnets were determined in maternal and fetal blood at 37 C. Halothane, isoflurane, diethyl ether, and nitrous oxide were significantly more soluble in maternal than in fetal blood, while methoxyflurane, fluroxene, and cyclopropane were significantly less soluble. Reasons for these differences cannot be accounted for by differences in the type or amount of hemoglobin present.