Particulate bioluininescence in dinoflagellates: dissociation and partial reconstitution.

With the same extraction conditions used for Gonyaulax polyedra, soluble and particulate bioluminescence can be isolated from two additional species, Pyrodinium bahamense and Pyrocystis lunula. We have been able, for all three species, to dissociate soluble luciferin and luciferase from the particulate system. Luciferin can be incorporated into both reacted and unreacted particulate systems.

Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency.

Clinical applications of gene therapy mainly depend on the development of efficient gene transfer vectors. Large DNA molecules can only be transfected into cells by using synthetic vectors such as cationic lipids and polymers. The present investigation was therefore designed to explore the physicochemical properties of cationic lipid-DNA particles, with plasmids ranging from 900 to 52 500 bp. The colloidal stability of the lipoplexes formed by complexing lipopolyamine micelles with plasmid DNA of various lengths, depending on the charge ratio, resulted in the formation of three domains, respectively corresponding to negatively, neutrally and positively charged lipoplexes. Lipoplex morphology and structure were determined by the physicochemical characteristics of the DNA and of the cationic lipid. Thus, the lamellar spacing of the structure was determined by the cationic lipid and its spherical morphology by the DNA. The main result of this study was that the morphological and structural features of the lipopolyamine-DNA complexes did not depend on plasmid DNA length. On the other hand, their gene transfer capacity was affected by the size of plasmid DNA molecules which were sandwiched between the lipid bilayers. The most effective lipopolyamine-DNA complexes for gene transfer were those containing the shortest plasmid DNA.

Erythropoietin secretion and physiological effect in mouse after intramuscular plasmid DNA electrotransfer.

BACKGROUND: Direct intramuscular plasmid DNA injection has recently been proposed for erythropoietin therapy, as an alternative to either systemic injection of recombinant erythropoietin or the use of viral vectors for erythropoietin gene transfer. However, direct intramuscular plasmid injection has so far been hampered by low efficiency and high interindividual variability. METHOD: We explored the use of a new method termed 'intramuscular electrotransfer' for erythropoietin gene expression in the mouse. This method is based on intramuscular plasmid injection followed by application of appropriate electric pulses. RESULTS: Intramuscular plasmid electrotransfer in mouse leg led to an increase of approximately 10- to 100-fold in circulating murine erythropoietin level, as compared to naked DNA alone. Using electrotransfer, as little as 1 microgram of an erythropoietin encoding plasmid was sufficient to induce an increase in mouse hematocrit, from 47% up to 80%. This hematocrit increase was stable for at least two months. Moreover, interindividual hematocrit variability was markedly reduced by electrotransfer, as compared with naked DNA injection. CONCLUSION: In vivo electrotransfer appears to be a convenient method for obtaining high erythropoietin expression in mice, and it could also be used for the expression of other secreted therapeutic proteins.

Clinical, epidemiologic, and pulmonary function studies in alpha,-antitrypsin-deficient subjects of Pi Z type.

The results of pulmonary function testing and systematic medical history and epidemiologic data collection are reported for 20 persons with alpha 1-antitrypsin deficiency of Pi Z phenotype. The most common symptom, reported in 19 subjects (95 per cent), was dyspnea on exertion; 16 subjects (80 per cent) gave a history of wheezing, and 8 (40 percent) reported chronic cough and sputum production. The 8 women who had been pregnant reported a miscarriage rate of 29 per cent for all pregnancies. Respiratory symptoms and disease were commonly reported in the children of study subjects. Pulmonary function testing revealed abnormalities for 18 of 20 subjects, all of those 26 or more years of age. The test that was most frequently abnormal was the 1-sec forced expiratory volume expressed as a per cent of the forced vital capacity. All pulmonary function studies demonstrated a trend toward increased impairment with increased age, which was evident by the fourth decade. Within this group of persons having severe alpha1-antitrypsin deficiency, there was no correlation between serum concentrations of antitrypsin and subjective or objective indices of pulmonary disease. A group of 7 subjects who were incidentally found to have Pi Z alpha1-antitrypsin deficiency exhibited symptoms and pulmonary function abnormalities comparable to those of 13 subjects who were originally referred for known or suspected pulmonary disease. These data suggest that if interventions such as smoking cessation and occupational counseling are to be effective, they should be initiated before the fourth decade of life.

Production of a new DNA vehicle for gene transfer using site-specific recombination.

Supercoiled DNA molecules, minicircles, were produced by in vivo site-specific recombination. They contained exclusively the desired excisable fragment. Recombination was driven by bacteriophage lambda integrase from a plasmid substrate containing the attP and attB recombination sites in the same orientation. Conditions for minicircle production within the lysogen Escherichia coli D1210HP were optimised. Up to 1.5 mg minicircles could be produced per litre bacterial culture, and the remaining, unrecombined plasmid comprised less than about 15% of the minicircle produced. However minicircle multimers were also produced, and comprised up to 30% of all minicircles synthesised. The par ABCDE' locus from plasmid RK2 was introduced into the minicircle fragment, resulting in minicircle dimers being reduced to less than 2% of all minicircles. The par A gene encodes a resolvase that catalyses recombination at the multimer resolution site in the parABCDE' locus. Minicircle multimers were also resolved when par A was introduced downstream from the integrase gene of the lambda pL transcript in D1210HP together with a multimer resolution site carried by the minicircle fragment.

Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer.

Minicircles are a new form of supercoiled DNA molecule for nonviral gene transfer which have neither bacterial origin of replication nor antibiotic resistance marker. They are thus smaller and potentially safer than the standard plasmids currently used in gene therapy. They were obtained in E. coli by att site-specific recombination mediated by the phage lambda integrase, which was used to excise the expression cassette from the unwanted plasmid sequences. We produced two minicircles containing the luciferase or beta-galactosidase gene under the control of the strong human cytomegalovirus immediate-early enhancer/promoter. Comparing maximal differences, these minicircles gave 2.5 to 5.5 times more reporter gene activity than the unrecombined plasmid in the NIH3T3 cell line and rabbit smooth muscle cells. Moreover, injection in vivo into mouse cranial tibial muscle, or human head and neck carcinoma grafted in nude mice resulted in 13 to 50 times more reporter gene expression with minicircles than with the unrecombined plasmid or larger plasmids. Histological analysis in muscle showed there were more transfected myofibers with minicircles than with unrecombined plasmid.

Chronic obstructive pulmonary disease and alpha-1-antitrypsin (Pi) variation: a family study.

A kindred of a white male proband with alpha1-antitrypsin deficiency, ZZ phenotype, and a severe obstructive ventilatory defect secondary to pulmonary emphysema was studied with regard to alpha1-antitrypsin phenotype, serum trypsin inhibitory capacity (T.I.C.), and pulmonary function. T.I.C.'s and alpha1-antitrypsin phenotypes were consistent with autosomal codominant inheritance. While MZ relatives as well as the proband had pulmonary function abnormalities, the pattern varied within the kindred and abnormalities were observed in some MM relatives also.

Risk factors in chronic obstructive pulmonary disease (COPD).

In a genetic-epidemiologic study of chronic obstructive pulmonary disease (COPD) observations adjusted for age, sex, race, and smoking indicate certain factors to be associated with increased pulmonary function aberrancy, and suggest that they are risk factors for COPD. These presumptive "risk factors" include not only cigarette smoking, but also alpha1-antitrypsin (Pi system) variation, one or more other familial components, low socioeconomic status (SES), and, in whites, ABO blood type (either absence of "B" or presence of "A").

A common familial component in lung cancer and chronic obstructive pulmonary disease.

First-degree relatives of lung-cancer patients and of patients with chronic obstructive pulmonary disease had significantly higher age-sex-race-smoking-adjusted rates of impaired forced expiration than first-degree relatives of patients with non-pulmonary disease or community-derived comparison series (neighbourhood controls and teachers). Subclassification of the data and multiple adjustment for smoking, race, sex, and other confounding factors emphasised the consistency of the pattern. These findings strongly suggest that lung cancer and chronic obstructive pulmonary disease share a common familial component other than smoking. The clinical manifestation may depend on the presence of one or more other cofactors as yet undefined.

A genetic-epidemiologic study of chronic obstructive pulmonary disease. I. Study design and preliminary observations.

Although certain environmental agents (e.g., cigarette smoking) are known to be causally related to chronic obstructive pulmonary disease (COPD), differential response to their deleterious effects suggests the importance of constitutional (host) factors. The voluminous literature on the familial occurrence of COPD as well as the association between genetically determined serum alpha 1-antitrypsin (alpha 1-at) deficiency and COPD, however, reveals many aspects yet to be clarified. Studies of alpha 1-at indicate that neither its nature nor its relationship to COPD is simple. Moreover, other familial factors are likely involved. To obtain a better understanding of the etiology and pathogenesis of COPD, both genetic and environmental factors are being examined in a multifaceted investigation. The preliminary observations are summarized. COPD patients have a higher frequency of Pi variant phenotypes than those without lung disease. Among other subjects, both cigarette smoking and Pi variant phenotypes are associated with increased pulmonary function abnormality. Finally, there is familial aggregation of pulmonary impairment that cannot be explained entirely by Pi type or smoking.