Advances in alternative DNA delivery techniques

This review describes recent advances in alternative DNA-delivery techniques with particular emphasis on silicon carbide fibers, intact tissue electroporation, electrophoresis and microinjection. The advantages/disadvantages of each method along with a historical overview and theory of practice are discussed.     

Differential metabolism of sodium azide in maize callus and germinating embryos

Sodium azide is a potent mutagen of maize (Zea mays L.) kernels that may have potential as a point mutagen for inducing biochemical mutations in maize tissue cultures. Azide mutagenicity was evaluated in friable, embryogenic maize callus and a nonregenerable maize suspension culture by determining the number of resistant variant cell lines able to grow on media containing inhibitory concentrations of lysine plus threonine (LT). The number of LT-resistant variants selected from either culture type did not increase in response to azide treatment. In addition, there was no increase in somatic mutations in more than 100 plants regenerated from azide treated LT-resistant lines. The levels of mutagenic metabolite of azide (presumably azidoalanine), were determined by bioassay in the two azide-treated maize callus types and compared to levels of mutagenic metabolite in embryos isolated from azide-treated kernels. The two types of maize tissue cultures and isolated embryos contained similar levels of mutagenic metabolite 4 h after azide treatment indicating similar uptake and conversion of azide to mutagenic metabolite in the three tissues. Mutagenic metabolite in azide-treated embryos did not significantly decrease after 40 h. However, mutagenic metabolite levels in both azide-treated tissue cultures decreased to near background levels within 20 h providing evidence for rapid metabolism of the azide mutagenic metabolite. The lack of evidence for azide mutagenicity in maize callus and its known potent mutagenicity in kernels appears to be associated with specific differences in azide metabolism between callus tissues and kernel embryos.     

Inability of murine peritoneal macrophages to convert linoleic acid into arachidonic acid. Evidence of chain elongation

Various murine macrophage populations synthesize and secrete large amounts of arachidonic acid (20:4n-6) derived eicosanoids (cyclo-oxygenase and lipoxygenase products). These metabolites are known to possess a wide variety of functions with regard to the initiation and regulation of inflammation and tumorigenesis. Because the dietary intake of 20:4n-6 is usually low, tissues are largely dependent upon dietary linoleic acid (18:2n-6) as an initial unsaturated precursor for the biosynthesis of 20:4n-6. The purpose of these experiments was to determine whether resident or responsive murine macrophages possess desaturase and elongase activities capable of in vitro conversion of 18:2n-6 into 20:4n-6. Peritoneal exudate macrophages were purified by adherence and incubated in serum-free medium containing fatty acid-free BSA with [1-14C] 18:2n-6. Approximately 90 to 98% of the [14C]18:2n-6 at 4 and 16 h was recovered in phosphatidylcholine and phosphatidylethanolamine. The metabolism of [14C]18:2n-6 was determined after transesterification and separation of the 14C-fatty acid methyl esters by argentation TLC, reverse phase HPLC, and electron impact gas chromatography/mass spectrometry. Resident and responsive macrophages lacked the capacity to transform [14C]18:2n-6 into 20:4n-6. In addition, prelabeled macrophages incubated with soluble, calcium ionophore A23187 or phorbol myristate, or particulate, zymosan, membrane perturbing agents also lacked delta 6 desaturase activity. All macrophages tested were capable of elongating [14C]18:2n-6 into [14C]20:2n-6. These observations suggest that 20:4n-6, present in macrophage phospholipids, is biosynthesized elsewhere and transported to the macrophage for esterification into the phospholipids. In addition, these findings demonstrate that elongase activity is present in both the resident and responsive peritoneal macrophage.     

Hormone-induced vocal behavior and midbrain auditory sensitivity in the green treefrog,Hyla cinerea

Summary Twenty four castrated male, 6 intact male, and 11 intact female Hyla cinerea were injected subcutaneously with 25 g arginine-vasotocin (AVT) and induced to call 1 h later in response to the playback of a conspecific mating call. Eighteen castrated males and 8 intact females were implanted 5 mg androgen pellets for 3 weeks prior to the neuropeptide injection. Among castrated males, 6/9 testosterone (T) implanted, 4/9 dihydrotestosterone (DHT) implanted and 2/6 non implanted individuals produced calls after being administered AVT. 5/6 intact non implanted males and 6/8 T intact implanted females also called, and 3 intact non implanted females remained silent after the injection. Evoked calls had a mid-frequency spectral peak at about 1900 Hz which is absent in field-recorded mating calls of this species. Calls of implanted females and castrated non implanted males were shorter than those of castrated implanted and intact non implanted males. Audiograms measured before hormone implants showed dips of enhanced sensitivity at about 0.5, 0.9 and 3.0 kHz in males and females. After AVT injection, thresholds at frequencies within the 0.7–1.5 kHz range were increased in castrated males. Such reduction in sensitivity points to an inhibition of the auditory system during hormone induced vocal activation.Abbreviations AVT arginine-vasotocin - DHT dihydrotestosterone - T testosterone - TS torus semicircularis     

Comparison of transforming growth factor β and a human tumour-derived suppressor factor

Summary Serum-free supernatants from the human melanoma cell line G361 contain a factor that can potently suppress the generation of tumouricidal lymphokine-activated killer (LAK) cells in response to interleukin-2. To characterise the suppressive factor of tumour origin we performed a number of physicochemical and functional comparisons with another immunosuppressive protein, transforming growth factor (TGF). The bioactivity of tumour-derived suppressor factor (TDSF), assayed by suppression of LAK cell generation, was unaffected by a reducing agent but lost when denatured with a chaotropic agent. In contrast, TGF was inactivated by reduction but not denaturation. TDSF lost bioactivity in conditions of pH less than 4, whereas TGF showed no loss of activity. The TDSF moiety has an estimated pI of 4.3 and a molecular mass of 69–87 kDa. This differs from published values of pI 9.5, and 25 kDa molecular mass for TGF. Anti-TGF antiserum reversed the effects of TGF but did not affect the suppression of LAK cell generation caused by TDSF. These findings provide compelling evidence that the TDSF moiety is not TGF, and may be a novel immunoregulatory cytokine.     

Isolation of histamine-producing Lactobacillus buchneri from Swiss cheese implicated in a food poisoning outbreak.

A histamine-producing strain of Lactobacillus buchneri was isolated from Swiss cheese that had been implicated in an outbreak of histamine poisoning. It produced up to 4,070 nmol of histamine per ml in MRS broth supplemented with 0.1% histidine. The identification of this isolate was based on its biochemical, bacteriological, and DNA characterizations.