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Biomedical subjects

M J Walker

Publications and source records attributed to M J Walker.

At least 127 records · Page 7Linked to original sources
PubMed1991

Construction of minitransposons for constitutive and inducible expression of pertussis toxin in bvg-negative Bordetella bronchiseptica.

Appropriately detoxified pertussis toxin (PT) of Bordetella pertussis is considered to be an essential component of new-generation whooping cough vaccines, but the development of a procedure to obtain high levels of purified toxin has been and continues to be a major difficulty. To produce a system enabling the biological separation of PT from other virulence determinants of B. pertussis and the attainment of high yields of the toxin, minitransposons containing the PT operon were constructed and stably integrated into the chromosome of Bordetella virulence regulatory gene (bvg)-negative Bordetella bronchiseptica ATCC 10580. Since the minitransposons introduced into Bordetella spp. lack the cognate transposase function, they are unable to undergo further transposition events or mediate gene deletions and rearrangements that lead to strain instability. The TnPtacPT minitransposon contains the PT operon under the control of the tac promoter and directs IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible expression of PT in B. bronchiseptica ATCC 10580. The level of IPTG-induced PT expression was, however, lower than that found for the wild-type B. pertussis Tohama I strain. The TnfusPT minitransposon contains a promoterless PT operon which is only expressed after insertion of the transposon downstream of an appropriately oriented indigenous promoter. After "promoter probing" of B. bronchiseptica with the transposon, clones were screened for PT production by immunoblotting with specific monoclonal antibodies. One clone, designated B. bronchiseptica 10580:: TnfusPT1, expresses significantly higher levels of PT than does B. pertussis Tohama I. The recombinant toxin produced was biologically active in the Chinese hamster ovary cell-clustering assay. High-level expression of PT from a B. bronchiseptica host promoter should provide better yields of the toxin from bacteria not producing other bvg-regulated pathogenesis factors that may play a role in the undesired side effects of current pertussis vaccine preparations.

Bordetella bronchiseptica↗
PubMed1991

Characterization of murine monoclonal antibodies that recognize defined epitopes of pertussis toxin and neutralize its toxic effect on Chinese hamster ovary cells.

Three murine monoclonal antibodies (MAb), E19, E205, and E251, raised against pertussis toxin reacted in Western blots (immunoblots) with the S1, S4, and S2-S3 subunits, respectively, and neutralized the Chinese hamster ovary cell-clustering activity of pertussis toxin. MAb E251 recognized a linear synthetic peptide corresponding to amino acids 107 to 120 of the S2 subunit, suggesting a role for this region in receptor binding.

Animals↗
PubMed1991

Antibody responses in the lungs of mice following oral immunization with Salmonella typhimurium aroA and invasive Escherichia coli strains expressing the filamentous hemagglutinin of Bordetella pertussis.

The filamentous hemagglutinin (FHA) of Bordetella pertussis was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, and in a strain of Escherichia coli harboring Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness for epithelial cells. Expression of FHA in these strains did not interfere with their ability to invade Henle cells. Immunoglobulins A and G specific for FHA were detected in lung washes of mice following oral immunization with the live recombinant organisms; antibody levels were significantly higher than those in mice immunized with killed bacteria administered orally or intraperitoneally. Live oral vaccines carrying protective antigens of B. pertussis may be an important alternative to new-generation component vaccines against whooping cough.

Adhesins, Bacterial↗
PubMed1991

Production of recombinant Bordetella pertussis serotype 2 fimbriae in Bordetella parapertussis and Bordetella bronchiseptica: utility of Escherichia coli gene expression signals.

Serotype-specific fimbriae of Bordetella pertussis are considered potential components of new-generation vaccines against whooping cough. Attempts to characterize fimbriae, and indeed other virulence determinants, produced by B. pertussis have been frustrated on one hand by low yields from B. pertussis itself and on the other by an inability to produce native recombinant products in Escherichia coli. In order to try to circumvent this problem, we have examined the expression of B. pertussis serotype 2 fimbriae in Bordetella parapertussis and Bordetella bronchiseptica from native as well as E. coli expression signals. These studies revealed that the fimbrial gene product was expressed from the original B. pertussis promoter and Shine-Dalgarno sequence in both B. parapertussis and B. bronchiseptica. The transcriptional start site of the gene was located 146 nucleotides upstream of its ATG start codon. A recombinant fimbrial subunit gene containing PLAC and the atpE translation initiation region of E. coli was also expressed in B. bronchiseptica. In all cases in which gene expression was detected the gene product was expressed as serotype 2-specific fimbriae as determined by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopic investigation of the bacterial cell surface.

Base Sequence↗
PubMed1991

Tissue distribution of mexiletine enantiomers in rats.

1. The kinetics of distribution of the enantiomers of mexiletine were studied in various tissue (heart, brain, lungs, liver, kidneys and fat) in male Sprague-Dawley rats after administration of a single i.v. dose (10 mg/kg) of racemic mexiletine. 2. The pharmacokinetic parameters calculated from the serum data showed a 32% greater systemic clearance (162 ml/min per kg vs 123 ml/min per kg) and a 22% greater steady-state volume of distribution (9.0 l/kg vs 7.4 l/kg) for R(-)-mexiletine relative to the S(+)-enantiomer. However, the terminal elimination half-lives of the enantiomers (1.4 and 1.3 h for R(-)- and S(+)-mexiletine, respectively) did not exhibit stereoselectivity. 3. Maximum tissue concentrations of the enantiomers were observed at 5 min after dosage in all tissues studied. Stereoselective uptake was evident only in the liver tissue and was 2.4-fold greater for S(+)-mexiletine. High tissue/serum ratios (greater than 20 for both enantiomers) were observed in lungs, brain and kidneys. The cardiac concentrations of R(-)- and S(+)-mexiletine were 8- and 7-fold those of serum, respectively. 4. The results demonstrate that the uptake of mexiletine enantiomers into the target tissue (heart) is not stereoselective. However, the relatively high brain accumulation of the enantiomers may be related to the CNS side-effects commonly associated with mexiletine therapy.

Animals↗
PubMed1991

Actions of platelet-activating factor on isolated rat hearts.

The actions of Platelet-Activating Factor (PAF) on isolated rat hearts were investigated. In a dose-dependent manner PAF decreased peak systolic pressure and maximum rate of rise of intraventricular pressure (+dP/dt(max]. PAF dose-dependently decreased coronary flow, prolonged the P-R interval of the EKG, and decreased heart rate. These actions of PAF were only partially blocked by drugs blocking receptors for PAF (CV 3988, WEB 2086), thromboxanes (ONO 3708), or leukotrienes (FPL 55712, L-655,240) or by a blocker of eicosanoid production (ibuprofen). Since depressed contractions will of themselves reduce coronary flow, PAF's action on flow was also investigated in hearts whose contractions were blocked by elevated potassium concentrations plus tetrodotoxin. Such treatment did not prevent PAF reducing coronary flow, indicating that a direct vasoconstriction occurred. The reductions in coronary flow in non-contracting hearts induced by PAF were equal to those induced by the coronary vasoconstrictors vasopressin and ergonovine under the same conditions. In order to isolate direct effects of PAF on myocardial contractile cells from effects mediated via changes in coronary flow, PAF was given to hearts maximally dilated by a concentration of nifedipine (0.03 microM) which had no effect on contractility. This concentration of nifedipine increased flow from 15.5 +/- 1.4 mL/min to 18.1 +/- 2.4 mL/min. In the presence of this nifedipine-induced vasodilation, PAF still exhibited negative inotropic actions, but without reducing coronary flow. Thus the effects of PAF on isolated rat hearts involve direct actions on both myocardial contracting cells and on coronary vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Results of treatment of stage I-III breast cancer in black Americans. The Cook County Hospital experience, 1973-1987.

Whether the prognosis for black women with breast cancer differs from that of nonblack women remains controversial. The treatment results of 526 black women who received definitive therapy for Stage I-III breast cancer at Cook County Hospital, 1973 through 1987 are presented. The 5-year and 10-year projected survival rates for 272 node-negative patients (83.9% and 76.6%, respectively) and for 72 node-positive nonadjuvant treated patients (58.1% and 35.2%, respectively) are similar to those reported in the literature for nonblack patients. Adjuvant therapy improved the projected relapse-free (P = 0.0744) and overall survival curves (P = 0.0448) for 182 node-positive patients compared with nonadjuvant patients. The greatest benefit was seen for patients greater than 50 years of age with one to three positive nodes. The incidence of estrogen and progesterone receptors was found to be similar to those reported for nonblack patients. Once breast cancer has been diagnosed and appropriately treated, there appear to be few differences in the natural history of breast cancer between black and nonblack patients.

Adenocarcinoma↗
PubMed1990

Characterization of malignant mesenchymal cell line (UISO-RS-3) derived from a human rhabdomyosarcoma and inhibition by pharmacologic doses of estrogen.

A new tumor cell line has been established from a malignant pleural effusion in a 28-yr-old female patient with a primary alveolar rhabdomyosarcoma of the left buttock. The in vitro and in vivo growth characteristics, morphologic features, abnormal karyotype, and immunohistochemical staining pattern indicate that this cell line is comprised of primitive malignant mesenchymal cells derived from a human rhabdomyosarcoma. Receptor studies done on tumors grown in male athymic mice revealed a single class of high affinity saturable cytoplasmic estrogen receptor (Bmax 2.6 fm/mg cytosol protein, Kd 0.34 nM). Likewise, sucrose density gradient analysis demonstrated specific low-capacity, high-affinity estradiol binding predominately in the 8S region. Cell growth in monolayer culture and on soft agar in the presence of estradiol was inhibited by pharmacologic concentrations of estradiol in a dose-responsive manner compared with control. We describe a newly characterized malignant mesenchymal cell line derived from an alveolar rhabdomyosarcoma that is inhibited by pharmacologic doses of estradiol in vitro. These findings suggest further investigation into the mechanism(s) of this estrogen-induced inhibition in rhabdomyosarcomas.

Adult↗
PubMed1990

Parker and Radow test of drug withdrawal aversion: opposite effect in rats chronically infused with sufentanil or amphetamine.

In rats, cessation of periodic injections of morphine reduces a preference for a palatable saccharin solution presented in a choice with water, and this has been interpreted to reflect withdrawal malaise. We confirmed and examined this "Parker and Radow Model" using subcutaneously implanted osmotic minipumps as the means of drug delivery and the opiate, sufentanil, and the psychostimulant, amphetamine, as the treatment drugs; surgical removal of the pumps was used to initiate withdrawal. Thus, rats withdrawn after 2 weeks exposure to a sufentanil-delivering pump (0.25 microgram/hr) showed a decreased preference for the saccharin and animals exposed to an amphetamine pump (68 micrograms/hr) showed an increased preference, as compared to placebo-exposed controls. This pattern of effects was systematically replicated in new subjects using 4 weeks of treatment and 136 micrograms/hr amphetamine. Since the locomotor increasing and body weight decreasing effects of amphetamine were also demonstrated and the doses of amphetamine and sufentanil were in comparable dose ranges, it was concluded that the Parker and Radow procedure may be a reliable measure of opiate withdrawal, but under similar test and treatment conditions other processes may be operative in amphetamine-treated animals. Problems of measuring motivation of withdrawal, particularly of spontaneous withdrawal, were noted.

Analgesics, Opioid↗
PubMed1990

Malignant melanoma of the female genitalia.

Fifteen women with genital malignant melanoma were studied. Their ages ranged from 19 to 66 years (mean 49.1 years); 12 were white, and three were black. The sites of involvement were the mons pubis (one patient), perineal body (one), labium majus (three), labium minus (three), and vagina (seven). Ten patients (66.6%) died of their disease, one is alive with disease, and four are alive without evidence of disease. For the living patients the duration of follow-up was 20 to 118 months (mean 63.6 months). Of those who died, survival ranged from 3 to 76 months (mean 25.1 months). The predominant type of malignancy was superficial spreading melanoma 50%. Nodular melanoma represented 22%, and the nodular polypoidal variant 14%. Melanoma of the squamous mucosa, also referred to as lentiginous melanoma, constituted 14%. By using Chung's method of determining levels of invasion, we found that no lesion was in situ (level I), two were level II (less than 1.0 mm thick), one was level III (between 1.0 and 2.0 mm) and the remaining 11 patients had lesions that were greater than 2.0 mm (levels IV and V). Because the subcutaneous fat is not consistently present in all sites of the female genitalia, all tumors thicker than 2.0 mm were included in level IV, and no level V tumors were classified in our study. Using Breslow's microstaging method, we found the thickness to range from 0.65 to 9.5 mm (mean 4.75 mm). When we correlated survival with level and thickness of tumor in nine patients who died, one tumor was level III and eight were level IV; thickness ranged from 1.65 to 9.0 mm (mean 5.64 mm).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Engineering upstream transcriptional and translational signals of Bordetella pertussis serotype 2 fimbrial subunit protein for efficient expression in Escherichia coli: in vitro autoassembly of the expressed product into filamentous structures.

Escherichia coli containing a cloned gene encoding the Bordetella pertussis serotype 2 fimbrial subunit failed to produce detectable levels of the gene product in whole-cell extracts. To engineer plasmids capable of directing the expression in E. coli of high levels of this product, both as a pre-protein and as a methionylated mature form the upstream signals of the fimbrial subunit gene were replaced by the lambda P(L) and P(R) promoters and the E. coli atpE translational initiation region. These constructs did not result in the expression of fimbrial subunit at detectable levels in several E. coli strains including DH5. However, they did in E. coli CAG629, which is lon protease and heat shock protein deficient. Both pre-protein and methionylated mature protein had molecular weights of 25.0 kD, which indicated that correct processing of the leader sequence had occurred and thus that it was transposed across the inner membrane. Electron microscopic investigation of the cell surface of E. coli cells expressing either form of the fimbrial gene failed to detect the presence of filamentous structures. The methionylated mature form of the recombinant fimbrial subunit was purified to apparent homogeneity. After dialysis in appropriate conditions it was seen to autoassemble into protein polymers. Antibodies raised against polymerized recombinant subunit reacted weakly with whole B. pertussis serotype 2 fimbriae in immunodot blot assays. However, such antibodies reacted in Western blots equally well with the recombinant and wild-type form of the fimbrial subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Bacterial↗
PubMed1990

Effects of verapamil, nifedipine and flunarizine on haemodynamics and regional blood flows in pentobarbitone-anaesthetized rats.

Representative calcium antagonists from proposed class I (nifedipine), class II (verapamil) and class III (flunarizine) have been examined for effects on blood pressure, heart rate, ventricular pressures, ECG, cardiac output and regional blood flow in pentobarbitone-anaesthetized rats. Flow was measured by the microsphere technique. Low and high infusion rates of each drug were chosen to decrease mean arterial pressure by 25 and 40 mmHg, respectively. At equi-depressor infusion rates, all drugs equally decreased total peripheral resistance and slightly increased cardiac output and stroke volume. Heart rate was decreased by verapamil and flunarizine, but increased by nifedipine. Verapamil markedly decreased dP/dtmax of ventricular pressure and prolonged the PR-interval. Flunarizine was similar. Nifedipine decreased dP/dtmax and had no effect on the PR-interval of the ECG. Similar effects on regional blood flow were seen with the three drugs; flow to lungs, heart, liver, skeletal muscle and stomach increased. Correction for blood pressure changes, i.e. conductance, showed that vasodilatation occurred in all regions, with all drugs, except for the skin. Therefore, representatives from three sub-classes of calcium antagonists had similar effects on blood flow but different effects on the heart.

Anesthesia↗
PubMed1990

Attitudes are contagious. What are you spreading around?

1. High scrutiny, rapid change, and demands for higher levels of production and performance produce stress and uncertainty, which can exact a severe toll on individual morale, as well as the efficiency of a work unit or entire organization. 2. Negative rumors are spread when needed information is limited, and when individuals are anxious, frustrated, hurt, or angry; each person either "poisons the well" or offers encouraging antidotes. 3. Those who learn active listening, accompanied by insightful understanding and compassion, are the organization's healers, helpers, and positive leaders.

Attitude of Health Personnel↗
PubMed1989

The influence of glucocorticoids on the growth of a human leiomyosarcoma cell line SK-LMS-1.

A cell line derived from a human leiomyosarcoma, SK-LMS-I, has cystolic [Bmax 201.6 +/- 39.7, means +/- SD, fm/mg cytosol protein KD 14.6 +/- 7.8 nM (n = 8)] and nuclear [Bmax 189.2 +/- 87.3 fM/mg DNA, KD 4.8 +/- 0.26 nM (n = 5)] glucocorticoid receptor, by Scatchard analysis of tumors grown in male athymic mice. Tumor growth of SK-LMS-I cells in male athymic mice is inhibited by daily s.c. injection of DEX 5 micrograms, DEX 25 micrograms, DEX 5 micrograms with 5 mu RU-486 and 5 micrograms RU-486. In sharp contrast, in vitro, glucocorticoid markedly stimulates the growth (as determined by cell number) of SK-LMS-I cells, principally at higher cell densities (days 10-21 of growth carried on over a 21- to 23-day period), the greatest stimulation being seen with DEX 10(-6) to 10(-8) M, and no stimulation being seen with DEX 10(-9) and 10(-10) M. In vitro, glucocorticoids with higher affinity for the GR stimulate growth, steroids with lower affinity inhibit growth. No alterations in cell-cycle distribution (percent G0/G1, S, or G2/m) could be found by flow cytometric analysis of glucocorticoid-stimulated asynchronously growing cultures. Single, isolated, untreated SK-LMS-I cells form colonies in soft agar with an efficiency of 1.78 +/- 0.10%. Pre-treatment of cells with DEX 10(-7) M increases this to 3.24 +/- 0.17%, while cells pre-treated with both DEX 10(-7) M RU-486 10(7) M form colonies with the same efficiency as untreated cells. Glucocorticoids have inhibitory effects on in vivo growth and stimulatory effects on in vitro growth of a GR-positive human leiomyosarcoma cell line.

Animals↗