Heterologous expression of a green fluorescent protein-pertussis toxin S1 subunit fusion construct disrupts calcium channel modulation in rat superior cervical ganglion neurons.

The fusion construct pEGFP-PTXS1 was assembled by ligating cDNA encoding the S1 subunit of Bordetella pertussis toxin (PTX) into the plasmid pEGFP-C1 (which codes for enhanced green fluorescent protein). Microinjection of pEGFP-PTXS1 (1-100 ng/microl) into the nucleus of dissociated rat sympathetic ganglion neurons resulted in functional expression as determined from the diffuse green fluorescence and disruption of norepinephrine-mediated N-type Ca2+ channel modulation. The heterologously expressed toxin retained specificity for G alpha(i/o)-dependent pathways as VIP-mediated modulation of N-type Ca2+ channels and muscarine-mediated inhibition of M-type K+ channels persisted in pEGFP-PTXS1 expressing neurons. These data demonstrate that the S1 subunit of PTX is readily expressed in mammalian neurons and remains functional following fusion to the C-terminus of another protein.

Antibodies to the chlamydial 60 kilodalton heat shock protein in women with tubal factor infertility.

INTRODUCTION: Fallopian tube damage and subsequent infertility are common sequelae of upper genital tract infection with Chlamydia trachomatis. This fallopian tube damage is thought to be immune mediated. The 60 kilodalton chlamydial heat shock protein (hsp) may be the key antigen associated with this pathogenic response. Our objective was to study the relationship between antibody response to 60 kilodalton chlamydial hsp and tubal factor infertility (TFI). SUBJECTS AND METHODS: Twenty-three women with TFI and 33 women with male factor infertility (controls) were studied. Tubal factor infertility was defined as infertility for one year with hydrosalpinx or distal tubal occlusion. Patients' sera were tested for antibodies to the chlamydial hsp using an enzyme-linked immunosorbent assay (ELISA). A stepwise logistic regression was performed by each patient's age, race/ethnicity, self-reported history of chlamydia infection, gonorrhea, or pelvic inflammatory disease (PID), history of ectopic pregnancy, and antibodies to the chlamydial hsp. RESULTS: Eighteen of the 23 women with TFI had a positive result on the hsp ELISA (78.6%) versus 23.4% of controls. Risk factors for TFI were a history of PID (P = 0.022), "nonwhite" race (P = 0.004), history of ectopic pregnancy (P = 0.027), and antibodies to the 60 kilodalton chlamydial hsp (P < 0.001). CONCLUSIONS: Antibodies to 60 kilodalton chlamydial hsp are strongly associated with TFI.

Tumor necrosis factor-alpha response to infection with Chlamydia trachomatis in human fallopian tube organ culture.

OBJECTIVE: Our purpose was to determine whether tumor necrosis factor-alpha is produced in response to infection with Chlamydia trachomatis in the fallopian tube. STUDY DESIGN: Fallopian tubes were harvested at the time of abdominal hysterectomy and processed by standard tissue culture techniques. Tubal segments were inoculated with Chlamydia trachomatis serotype E/UW-5/CX. At 48 hours of incubation supernatant fluid was assayed for tumor necrosis factor-alpha. Tubal segments were stained for chlamydial inclusions and tumor necrosis factor-alpha by use of immunohistochemical techniques. RESULTS: Mean tumor necrosis factor-alpha levels for infected segments were 92.1 +/- 21.3 pg/ml (mean +/- SEM) and for control segments were 61.9 +/- 13.9 pg/ml (p = 0.03 by paired t test). Tumor necrosis factor-alpha was predominantly localized in the tubal epithelium. CONCLUSIONS: Tumor necrosis factor-alpha is produced in response to chlamydial infection by the human fallopian tube. It is an important proinflammatory cytokine and may promote the production of other cytokines and immune-mediated damage of the fallopian tube.

The Social Service Department Archives: Peking Union Medical College 1928-1951.

The case records in the Social Service Department Archives of the Peking Union Medical College not only provide information on medical practices in China between 1928 and 1951, but also a wealth of information on the social and economic conditions in China at that time, particularly for women and families. The records also provide information on the philanthropic work of the Rockefeller Foundation, which provided vast amounts of grant funds for the Peking Union Medical College. Research in the archives is limited, however, by the large number of files and the lack of finding aids, as well as the ever-changing attitude of the hospital administration toward researchers' use of the archives.

The detection of adducts in human cervix tissue DNA using 32P-postlabelling: a study of the relationship with smoking history and oral contraceptive use.

32P-Postlabelling was used to measure DNA adducts in the human cervix. Adduct levels were compared with patient smoking histories and contraceptive use. DNA adducts were found in 43 out of 58 samples. The number of adducts ranged from 0.2 to 59.5 adducts/10(8) nucleotides, though no significant difference was found to exist between the number of DNA adducts detected and the smoking history of each patient. In contrast, a significant difference at the 1% probability level was found between the adduct levels obtained from the cervical DNA of smokers who had used oral contraceptives and smokers who did not. Autoradiograms revealed a variety of adduct patterns. Some were found to have a diagonal zone of radioactivity which migrated from the origin of the TLC plate. Other autoradiograms revealed the presence of additional adduct spots located in the upper regions of the TLC plate, whereas others revealed the presence of these adduct spots alone. The origin of the adduct spots located in the upper regions of the TLC plate is unknown.

Immunoaffinity concentration of human lung DNA adducts using an anti-benzo[a]pyrene-diol-epoxide-DNA antibody. Analysis by 32P-postlabelling or ELISA.

DNA, isolated from 15 human lung autopsy samples, was examined for the presence of polycyclic aromatic hydrocarbon (PAH) DNA adducts. Using the nuclease P1 modification of the 32P-postlabelling technique, between 1 and 12 adducts/10(8) nucleotides were detected prior to immunoconcentration. Autoradiograms from most of the samples revealed a diagonal smear of radioactivity consistent with complex mixture (cigarette smoking) DNA damage. The DNA samples were digested to oligonucleotides, made single-stranded and subsequently applied to immunoaffinity columns containing immobilised anti-benzo[a]pyrene (B(a)P)-7,8-diol-9,10-epoxide (BPDE) DNA polyclonal rabbit antibody. The material remaining bound to the column, in addition to that passing through, was analysed using both ELISA and 32P-postlabelling techniques. Column-bound adducts comprised between 0% and 78% of any particular sample. Immunoconcentration, followed by 32P-postlabelling of the material which had been bound to the column, revealed the presence of a number of discrete adduct spots in autoradiograms of the more heavily adducted samples. Sample DNA not retained by the columns was also analysed; the chromatographic pattern obtained was a dense zone of radioactive material migrating from the origin. This evidence suggests that the composition of PAH-DNA adducts found in human lung samples exhibits wide inter-individual variation.

High-level expression using baculovirus, purification, and characterization of a monomeric form of type II calmodulin-dependent protein kinase.

The type II calmodulin-dependent protein kinase is an oligomer existing in multiple isozymic forms. To facilitate investigations of the regulatory mechanisms of this complex enzyme, we have constructed a truncated, calmodulin-dependent monomer of the alpha subunit. The N-terminal enzyme fragment (alpha 315) was expressed at high levels in a baculovirus/insect cell expression system. The recombinant protein was purified chromatographically using DEAE-cellulose, calmodulin-Sepharose, and AffiGel blue, yielding 4 mg of kinase from 1.5 x 10(8) cells in 4 h. Characterization of the truncated kinase indicated that it is a monomer and that interactions of alpha 315 with calmodulin and substrates are indistinguishable from those observed for purified holoenzyme from rat brain. These results indicate that the baculovirus/insect cell expression system is well suited for producing alpha 315, a structurally simplified model of the type II calmodulin-dependent protein kinase.

Griseofulvin: a new look at an old drug.

Griseofulvin is the oral antifungal agent of choice for the treatment of dermatophytoses. This article reviews the history, pharmacokinetics, adverse reactions, and traditional therapeutic applications of griseofulvin. In addition, reports since 1960 of the use of the drug in the treatment of Raynaud's phenomenon, progressive systemic sclerosis, lichen planus, mycosis fungoides, herpes zoster, eosinophilic fasciitis, and molluscum contagiosum are discussed, noting the varying degree of therapeutic success.

Calcineurin-phospholipid interactions. Identification of the phospholipid-binding subunit and analyses of a two-stage binding process.

Photoaffinity labeling of calcineurin by 1,2-distearoyl-sn-glycero-3-phospho-N-(4-azido-3-[125I]iodo-2- hydroxybenzoyl)ethanolamine resulted in preferential labeling of its regulatory B subunit. Photolabeling of B was greatly enhanced by Ca2+ which further supports the hypothesis that the phospholipid-binding site of calcineurin is located on this Ca2(+)-binding subunit. Extending the time of incubation of calcineurin with the photoprobe prior to photolysis also elevated labeling of the B subunit, probably as a result of time-dependent changes in protein conformation. Support for these conformational changes was obtained when time-dependent preincubation of calcineurin with acidic phospholipids enhanced subsequent tryptic degradation of its B subunit. Activity measurements and analyses of the reversibility of phospholipid-binding provided evidence for a two-stage mechanism of calcineurin-phospholipid interactions. Initial binding of calcineurin to phospholipids is rapid, Ca2(+)-sensitive, reversible, and leads to stimulation of the phosphatase toward a number of its substrates. A subsequent slow phase strengthens the association and appears to correlate with the phospholipid-promoted conformational change of the B subunit; the corresponding time-dependent effects on enzymatic activity are, again, substrate-dependent.

Linear relationship between DNA adducts in human lung and cigarette smoking.

Human lung and bladder DNA has been isolated and purified from either surgical or autopsy specimens. Smoking history details were obtained from patients or their close relatives. Each DNA sample was investigated using the nuclease P1 digestion modification of the 32P-postlabelling procedure. Data are presented for 48 lung and 19 bladder specimens. The samples were subdivided into three groups for data analysis, viz. smokers, former smokers and nonsmokers. The mean adduct levels (adducts per 10(8) nucleotides) in lung samples were: [see text] The chromatographic pattern of bladder DNA adducts for smokers was similar to that for smokers' lung DNA, although less intense. Adduct levels in former smokers tended to be lower than in smokers, although loss of adducts appeared to require several years after cessation of smoking. These findings support a link between DNA adduct levels and cigarette smoking, for both the lung and the bladder. For the former tissue there was a strong linear correlation between adduct levels and the number of cigarettes smoked.

Kinetic mechanism of the type II calmodulin-dependent protein kinase: studies of the forward and reverse reactions and observation of apparent rapid-equilibrium ordered binding.

The kinetic reaction mechanism of the type II calmodulin-dependent protein kinase was studied by using its constitutively active kinase domain. Lacking regulatory features, the catalytic domain simplified data collection, analysis, and interpretation. To further facilitate this study, a synthetic peptide was used as the kinase substrate. Initial velocity measurements of the forward reaction were consistent with a sequential mechanism. The patterns of product and dead-end inhibition studies best fit an ordered Bi Bi kinetic mechanism with ATP binding first to the enzyme, followed by binding of the peptide substrate. Initial-rate patterns of the reverse reaction of the kinase suggested a rapid-equilibrium mechanism with obligatory ordered binding of ADP prior to the phosphopeptide substrate; however, this apparent rapid-equilibrium ordered mechanism was contrary to the observed inhibition by the phosphopeptide which is not supposed to bind to the kinase in the absence of ADP. Inspection of product inhibition patterns of the phosphopeptide with both ATP and peptide revealed that an ordered Bi Bi mechanism can show initial-rate patterns of a rapid-equilibrium ordered system when a Michaelis constant for phosphopeptide, Kip, is large relative to the concentration of phosphopeptide used. Thus, the results of this study show an ordered Bi Bi mechanism with nucleotide binding first in both directions of the kinase reaction. All the kinetic constants in the forward and reverse directions and the Keq of the kinase reaction are reported herein. To provide theoretical bases and diagnostic aid for mechanisms that can give rise to typical rapid-equilibrium ordered kinetic patterns, a discussion on various sequential cases is presented in the Appendix.

Autophosphorylation of the type II calmodulin-dependent protein kinase is essential for formation of a proteolytic fragment with catalytic activity. Implications for long-term synaptic potentiation.

Autophosphorylation plays an essential role in proteolytic activation of the type II calmodulin-dependent protein kinase (CaM kinase II). Limited proteolysis of CaM kinase II by trypsin, alpha-chymotrypsin, and Ca2+-stimulated neutral protease (calpain) yielded a catalytically active kinase fragment only when the holoenzyme was autophosphorylated prior to proteolysis. Slightly larger, inactive fragments were obtained from nonphosphorylated CaM kinase II, regardless of whether Ca2+/calmodulin or Mg2+/ATP were present or absent. The active fragment exhibited Ca2+/calmodulin-dependent kinase activity with kinetic parameters identical with those of the activated holoenzyme. The key autophosphorylation site of CaM kinase II was absent from the active fragment which indicates that proteolysis can effectively uncouple the activation state and Ca2+/calmodulin independence of the kinase from the action of phosphoprotein phosphatases. Because autophosphorylation exerts such a tight control over this irreversible process, proteolytic activation of CaM kinase II by intracellular proteases offers an attractive mechanism for prolonging the effects of Ca2+ at the synapse.

Bile-salt-activated lipase: effect on kitten growth rate.

Because of the presence of bile-salt-activated lipase in cat milk, the dependence of the kitten on bile-salt-activated lipase is anticipated for milk fat absorption. To test this hypothesis, we initiated a feeding experiment comparing the growth rate of kittens fed with formula with those fed with formula and supplemented with purified human milk bile-salt-activated lipase. The results indicated that the kittens fed formula with supplemental enzyme had a growth rate twice that of kittens fed with formula alone. This study also indicated that the kitten can be utilized as an animal model in the investigation of the functional role of bile-salt-activated lipase. In this study we also performed the partial characterization of cat milk protein and fat.

Computational and site-specific mutagenesis analyses of the asymmetric charge distribution on calmodulin.

Calmodulin's calculated electrostatic potential surface is asymmetrically distributed about the molecule. Concentrations of uncompensated negative charge are localized near certain alpha-helices and calcium-binding loops. Further calculations suggest that these charge features of calmodulin can be selectively perturbed by changing clusters of phylogenetically conserved acidic amino acids in helices to lysines. When these cluster charge reversals are actually produced by using cassette-based site-specific mutagenesis of residues 82-84 or 118-120, the resulting proteins differ in their interaction with two distinct calmodulin-dependent protein kinases, myosin light chain kinase and calmodulin-dependent protein kinase II. Each calmodulin mutant can be purified to apparent chemical homogeneity by an identical purification protocol that is based on conservation of its overall properties, including calcium binding. Although cluster charge reversals result in localized perturbations of the computed negative surface, single amino acid changes would not be expected to alter significantly the distribution of the negative surface because of the relatively high density of uncompensated negative charge in the region around residues 82-84 and 118-120. However, this does not preclude the possibility of single amino acid charge perturbations having a functional effect on the more intimate, catalytically active complex. The electrostatic surface of calmodulin described in this report may be a feature that would be altered only by cluster charge reversal mutations. Overall, the results suggest that the charge properties of calmodulin are one of several properties that are important for the efficient assembly of calmodulin-protein kinase signal transduction complexes in eukaryotic cells.

Correlation of DNA adduct levels in human lung with cigarette smoking.

Lung cancer is the most common cancer in men in the United Kingdom and the second most common in women, accounting for between 25 and 40% of all cancer deaths. Cigarette smoking is widely accepted as the major cause of lung cancer and linear relationships have been established between the number of cigarettes smoked and lung cancer risk. Although approximately 50 carcinogenic chemicals have been identified in cigarette smoke, a causal link between specific compounds and lung cancer has yet to be made. Studies on cigarette smokers' urine, blood and placenta have provided indications of carcinogen exposure, and although the presence of covalently-bound adducts in human DNA provides evidence of exposure to carcinogens, there have been no reports of systematic studies on the levels of DNA adducts in human lung. We report here, using the 32P-post-labelling technique, that cigarette smokers have higher adduct levels than non-smokers, that there is a linear relationship between adduct levels and daily or lifetime cigarette consumption, and that people who have given up smoking for at least five years have adduct levels similar to those of non-smokers.

Potential liability in utilization review.

Increasingly, utilization review activities arising out of the cost containment concerns of third-party payers and the business community are becoming a factor in the health care delivery process. The author focuses on the potential liability of the physician and the utilization review organization when a patient suffers injury following a disagreement about treatment and urges the physician to deliver necessary services according to his or her medical judgments, not the judgement of the utilization review organization.