Navigating the post-Dobbs landscape: ethical considerations from a perinatal perspective.

Restrictive abortion laws have impacts reaching far beyond the immediate sphere of reproductive health, with cascading effects on clinical and ethical aspects of neonatal care, as well as perinatal palliative care. These laws have the potential to alter how families and clinicians navigate prenatal and postnatal medical decisions after a complex fetal diagnosis is made. We present a hypothetical case to explore the nexus of abortion care and perinatal care of fetuses and infants with life-limiting conditions. We will highlight the potential impacts of limited abortion access on families anticipating the birth of these infants. We will also examine the legally and morally fraught gray zone of gestational viability where both abortion and resuscitation of live-born infants can potentially occur, per parental discretion. These scenarios are inexorably impacted by the rapidly changing legal landscape in the U.S., and highlight difficult ethical dilemmas which clinicians may increasingly need to navigate.

Essentials of Neonatal-Perinatal Medicine fellowship: part 2 - clinical education and experience.

This is the second article in a seven-part series in the Journal of Perinatology that aims to critically examine the current state of Neonatal-Perinatal Medicine (NPM) fellowship training from the structure and administration of a program, to the clinical and scholarly requirements, and finally to the innovations and future careers awaiting successful graduates. This article focuses on the current clinical requirements; recent changes to the clinical environment and their effect on learning; and additional challenges and opportunities in clinical education.

Measuring dynamic Eustachian tube function using tympanometry in a pressure chamber: the effect of nasal betahistine application.

OBJECTIVE: To assess the effect of topical betahistine on Eustachian tube function in subjectively abnormal subjects in a hyperbaric chamber. METHOD: Active and passive Eustachian tube function was examined using tympanometry in a pressure chamber. RESULTS: Active Eustachian tube function was tested against the negative middle ear pressure induced by increasing the chamber pressure to +3 kPa. One voluntary swallow decreased middle-ear pressure by a mean of 1.36 kPa. Passive Eustachian tube function was tested by measuring spontaneous Eustachian tube openings as the chamber pressure dropped from +10 kPa to ambient. Four distinct patterns of Eustachian tube behaviour were seen, three of which indicated Eustachian tube dysfunction. Betahistine had no positive effect on Eustachian tube opening, although previous animal studies had suggested a beneficial effect. CONCLUSION: Topical betahistine had no effect on Eustachian tube function. Combining a hyperbaric chamber with tympanometry proved ideal for evaluating Eustachian tube function.

Do-Not-Resuscitate Orders in the Neonatal ICU: Experiences and Beliefs Among Staff.

OBJECTIVES: Studies in adult patients have shown that do-not-resuscitate orders are often associated with decreased medical intervention. In neonatology, this phenomenon has not been investigated, and how do-not-resuscitate orders potentially affect clinical care is unknown. DESIGN: Retrospective medical record data review and staff survey responses about neonatal ICU do-not-resuscitate orders. SETTING: Four academic neonatal ICUs. SUBJECTS: Clinical staff members working in each neonatal ICU. INTERVENTIONS: Survey response collection and analysis. MEASUREMENTS AND MAIN RESULTS: Participating neonatal ICUs had 14-48 beds and 120-870 admissions/yr. Frequency range of do-not-resuscitate orders was 3-11 per year. Two-hundred fifty-seven surveys were completed (46% response). Fifty-nine percent of respondents were nurses; 20% were physicians. Over the 5-year period, 44% and 17% had discussed a do-not-resuscitate order one to five times and greater than or equal to 6 times, respectively. Fifty-seven percent and 22% had cared for one to five and greater than or equal to 6 patients with do-not-resuscitate orders, respectively. Neonatologists, trainees, and nurse practitioners were more likely to report receiving training in discussing do-not-resuscitate orders or caring for such patients compared with registered nurses and respiratory therapists (p < 0.001). Forty-one percent of respondents reported caring for an infant in whom interventions had been withheld after a do-not-resuscitate order had been placed without discussing the specific withholding with the family. Twenty-seven percent had taken care of an infant in whom interventions had been withdrawn under the same circumstances. Participants with previous experiences withholding or withdrawing interventions were more likely to agree that these actions are appropriate (p < 0.001). CONCLUSIONS: Most neonatal ICU staff report experience with do-not-resuscitate orders; however, many, particularly nurses and respiratory therapists, report no training in this area. Variable beliefs with respect to withholding and withdrawing care for patients with do-not-resuscitate orders exist among staff. Because neonatal ICU patients with do-not-resuscitate orders may ultimately survive, withholding or withdrawing interventions may have long-lasting effects, which may or may not coincide with familial intentions.

A GH receptor antisense oligonucleotide inhibits hepatic GH receptor expression, IGF-I production and body weight gain in normal mice.

Diabetic retinopathy and acromegaly are diseases associated with excess action of GH and its effector IGF-I, and there is a need for improved therapies. We have designed an optimised 2'-O-(2-methoxyethyl)-modified phosphorothioate oligodeoxynucleotide, ATL 227446, and demonstrated its ability to suppress GH receptor mRNA in vitro. Subcutaneous injections of ATL 227446 reduced GH receptor mRNA levels, GH binding activity and serum IGF-I levels in mice after seven days of dosing. The reduction in serum IGF-I could be sustained for over ten weeks of dosing at therapeutically relevant levels, during which there was also a significant decrease in body weight gain in antisense-treated mice relative to saline and mismatch control-treated mice. The findings indicate that administration of an antisense oligonucleotide to the GH receptor may be applicable to human diseases in which suppression of GH action provides therapeutic benefit.

Insulin-like growth factor binding protein-3 (IGFBP-3) localizes to and modulates proliferative epidermal keratinocytes in vivo.

BACKGROUND: The colocalization of insulin-like growth factor binding protein-3 (IGFBP-3) and IGF-I receptor (IGF-IR) in the basal/germinative layer of the epidermis suggests a key role in modulating epidermal homeostasis. OBJECTIVES: We aimed to clarify both the specific cellular localization and the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. METHODS: (i) Total RNA was isolated from fluorescence-activated cell sorted basal human keratinocyte subtypes [keratinocyte stem cells, transit amplifying keratinocytes (TA), postmitotic differentiating keratinocytes (PMD)], and real-time polymerase chain reaction analysis was used to determine the abundance of IGFBP-3 and IGF-IR mRNAs. (ii) An IGFBP-3 transgenic mouse model was then used to assess the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. Excess epidermal IGFBP-3 mRNA and protein was determined by in situ hybridization and immunohistochemistry, respectively. RESULTS: (i) The highest levels of IGFBP-3 mRNA were detected in TA keratinocytes, in contrast to IGF-IR mRNA levels which were highest in PMD keratinocytes. (ii) Elevated human IGFBP-3 mRNA and protein was confirmed in the epidermis of skin derived from transgenic mice. Excess IGFBP-3 reduced the relative percentage of proliferative keratinocytes (Ki67 positive) irrespective of skin location (belly, back and tail). Thus, in the epidermis, IGFBP-3 mRNA is highly expressed by proliferative keratinocytes (TA) and overexpression of IGFBP-3 inhibits keratinocyte proliferation. CONCLUSIONS: We conclude that in vivo IGFBP-3 ensures epidermal homeostasis via downregulation of keratinocyte proliferation, and thus modulates the early stages of keratinocyte differentiation.

Delayed fluorescence from the photosynthetic reaction center measured by electronic gating of the photomultiplier.

The decay of the delayed fluorescence (920 nm) of reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides R26 in the P(+)Q(A)(-) charge-separated state (P and Q(A) are the primary donor and quinone, respectively) has been monitored in a wide (100 ns to 100 ms) time range. The photomultiplier (Hamamatsu R3310-03) was protected from the intense prompt fluorescence by application of gating potential pulses (-280 V) to the first, third, and fifth dynodes during the laser pulse. The gain of the photomultiplier dropped transiently by a factor of 1 x 10(6). The delayed fluorescence showed a smooth but nonexponential decay from 100 ns to 1 ms that was explained by the relaxation of the average free energy between P* and P(+)Q(A)(-) changing from -580 to -910 meV. This relaxation is due to the slow protein response to charge separation and can be described by a Kohlrausch relaxation function with time constant of 65 micros and a stretching exponent of alpha = 0.45.

Antisense oligonucleotide treatments for psoriasis.

Antisense oligonucleotides are emerging as an exciting therapeutic strategy for treating skin diseases such as psoriasis. Potential antisense targets are proteins upregulated in psoriatic skin, in particular those associated with inflammation (intercellular adhesion molecule [ICAM]-1, IL-2 and -8), proliferation (insulin-like growth factor type I receptor [IGF-IR], epidermal growth factor) and hyperangiogenesis (vascular endothelial growth factor [VEGF]). Whereas topical application and subsequent penetration of large oligonucleotides into normal skin is problematic, the impaired barrier function of psoriatic lesions permits the uptake of antisense drugs. Studies to date indicate that topically applied antisense molecules can be delivered to target cells in the epidermis and dermis of psoriatic skin. Antisense-mediated suppression of target mRNA and protein has been demonstrated in models of human skin grafted to immunosuppressed mice and in hairless mouse models of skin inflammation. In a xenograft model of human psoriasis, treatment with repeated intradermal injections of IGF-IR antisense caused a normalisation of the epidermal hyperproliferation. This class of drug, therefore, holds much potential for the successful treatment of psoriasis in the clinical setting.

Epitope mapping and specificity of the anti-alpha-synuclein monoclonal antibody Syn-1 in mouse brain and cultured cell lines.

While alpha- and beta-synuclein largely overlap in their expression in the vertebrate brain, only alpha-synuclein accumulates in the fibrillar aggregates typical of Parkinson's disease. It is thus critical to have immunological reagents that distinguish between these two protein isoforms. The monoclonal antibody Syn-1 (Transduction Labs) has been frequently used for the specific detection of alpha-synuclein. In this report, the epitope for Syn-1 is localized within residues 91-99 of human alpha-synuclein. Sequence differences exist in this domain that account for the specificity of Syn-1 for alpha- versus beta-synuclein. However, Syn-1 also displays reactivity with additional species (approximately 45 kDa) in brain homogenates from both wild-type and alpha-synuclein null mice, indicating a potential for cross-reactivity with a protein species that is unrelated to alpha-synuclein in brain tissue or extracts.

The electric field generated by photosynthetic reaction center induces rapid reversed electron transfer in the bc1 complex.

The cytochrome bc(1) complex is the central enzyme of respiratory and photosynthetic electron-transfer chains. It couples the redox work of quinol oxidation and cytochrome reduction to the generation of a proton gradient needed for ATP synthesis. When the quinone processing Q(i)- and Q(o)-sites of the complex are inhibited by both antimycin and myxothiazol, the flash-induced kinetics of the b-heme chain, which transfers electrons between these sites, are also expected to be inhibited. However, we have observed in Rhodobacter sphaeroides chromatophores, that when a fraction of heme b(H) is reduced, flash excitation induces fast (half-time approximately 0.1 ms) oxidation of heme b(H), even in the presence of antimycin and myxothiazol. The sensitivity of this oxidation to ionophores and uncouplers, and the absence of any delay in the onset of this reaction, indicates that it is due to a reversal of electron transfer between b(L) and b(H) hemes, driven by the electrical field generated by the photosynthetic reaction center. In the presence of antimycin A, but absence of myxothiazol, the second and following flashes induce a similar ( approximately 0.1 ms) transient oxidation of approximately 10% of the cytochrome b(H) reduced on the first flash. From the observed amplitude of the field-induced oxidation of heme b(H), we estimate that the equilibrium constant for sharing one electron between hemes b(L) and b(H) is 10-15 at pH 7. The small value of this equilibrium constant modifies our understanding of the thermodynamics of the Q-cycle, especially in the context of a dimeric structure of bc(1) complex.

Effects of exposure to event 176 Bacillus thuringiensis corn pollen on monarch and black swallowtail caterpillars under field conditions.

The widespread planting of corn genetically modified to produce Bacillus thuringiensis endotoxin has led to speculation that pollen from these fields might adversely affect nearby nontarget lepidopterans. A previous study of Bt corn engineered with Monsanto event 810 failed to detect an effect of pollen exposure on the black swallowtail, Papilio polyxenes, in either the field or the laboratory. Here, we report results of a field study investigating the impact of exposure to pollen from a Bt corn hybrid containing Novartis event 176 on two species of Lepidoptera, black swallowtails and monarch butterflies, Danaus plexippus. Nearly half of the 600 monarch larvae died within the first 24 h; this and subsequent mortality was not associated with proximity to Bt corn and may have been due in part to predation. Survivorship of black swallowtails was much higher than that of the monarchs and was also independent of proximity to the transgenic corn. However, despite five rainfall events that removed much of the pollen from the leaves of their host plants during the experiment, we observed a significant reduction in growth rates of black swallowtail larvae that was likely caused by pollen exposure. These results suggest that Bt corn incorporating event 176 can have adverse sublethal effects on black swallowtails in the field and underscore the importance of event selection in reducing environmental impacts of transgenic plants.

Antisense oligonucleotides in cutaneous therapy.

Antisense oligonucleotides have been the subject of intense interest as research tools to elucidate the functions of gene products and as therapeutic agents. Initially, their mode of action was poorly understood and the biological effects of oligonucleotides were often misinterpreted. However, research into these gene-based inhibitors of cellular action recently has succeeded in realising their exciting potential, particularly as novel therapeutic agents. An emerging application of this technology is in cutaneous therapy. The demand for more effective dermatological drugs will ensure further development of antisense strategies in skin, with key issues being drug delivery, therapeutic target selection, and clinical applicability.

Site specific labeling of Rhodobacter sphaeroides reaction centers with dye probes for surface pH measurements.

Covalently bound pH sensitive dyes are an important tool for characterizing the proteolytic reactions of protein complexes that play key roles in biological energy transduction. Here we demonstrate the feasibility of this method for photosynthetic reaction centers (RCs) for the first time, by the highly selective attachment of two thiol reactive derivatives of fluorescein to the two H subunit cysteines of the photosynthetic RC from Rhodobacter sphaeroides R-26 The pK(a) shifts of the dyes upon binding to the protein and in response to high salt were measured, and interpreted based on the structure of the RC. 2-[(5-fluoresceinyl)aminocarbonyl]ethyl-methanethiosulfonate was attached to Cys H156 and fluorescein-5-maleimide to Cys H234. By following the absorption changes of bound fluorescein (500 nm), and those of the hydrophilic pH indicator 8-hydroxypyrene-1,3,6-tris-sulfonic acid (468 nm), the surface and bulk pH were monitored separately with less than 5% crosstalk. Flash-induced proton uptake and external calibrations by mixing with aliquots of acid were measured in different redox states of the RCs. The results indicate that the charge in the quinone acceptor complex after flash activation (primary quinone acceptor (Q(A))- or secondary quinone acceptor (Q(B))-) has no effect on the surface pH and potential in the vicinity of these two attachment sites, between pH 6.5 and 9. Application of the method to other surface locations is discussed.

Calcium regulates the expression of insulin-like growth factor binding protein-3 by the human keratinocyte cell line HaCaT.

The insulin-like growth factor (IGF) system is essential for epidermal homeostasis. Insulin-like growth factor binding protein 3 (IGFBP-3), a modulator of IGF action that also exhibits IGF-independent activity, is localized to selected keratinocytes in the basal epidermal layer and may thus contribute to keratinocyte differentiation. We have utilized the human keratinocyte cell line, HaCaT, to examine the effect of calcium on the regulation of components of the IGF system. Western ligand and northern blot analyses revealed secreted IGFBP-3 and IGFBP-3 mRNA were reduced by an elevation in calcium levels in the culture medium. At 1.0 and 1.2 mM CaCl2 culture conditions IGFBP-3 abundance was reduced to 36% +/- 1.6% and 26% +/- 7.1%, respectively, of that from cells grown at 0.03 mM CaCl2. IGFBP-3 mRNA levels in 0.7 mM and 1.2 mM CaCl2 were reduced to 46% +/- 17.4% and 24% +/- 4.6%, respectively, compared with IGFBP-3 mRNA levels at 0.03 mM CaCl2. The observed reduction of IGFBP-3 was not associated with IGFBP-3 proteolysis. In contrast IGF-I receptor protein and mRNA levels remained unchanged. The IGF-I stimulated proliferative response of HaCaT keratinocytes showed that under low (0.03 mM) and high (1.2 mM) CaCl2 conditions IGF-I at 100 and 1000 ng per ml similarly increased cell number 2.4- and 2.7-fold, respectively, with similar dose-response curves. HaCaT keratinocytes grown under medium (0.7 mM) and high (1.2 mM), but not low (0.03 mM), CaCl2 conditions for 21 d revealed an induction of profilaggrin mRNA, a marker of keratinocyte differentiation. These studies indicate that the exposure of HaCaT keratinocytes to elevated calcium levels is associated with a decline in IGFBP-3 but not IGF-I receptor levels. These findings suggest a potential mechanism for the distribution of IGFBP-3 in the epidermis, which may be involved in the process of keratinocyte differentiation.

Protein control of the redox potential of the primary quinone acceptor in reactioncCenters from Rhodobacter sphaeroides.

The role of the protein environment in determining the redox midpoint potential (E(m)) of Q(A), the primary quinone of bacterial reaction centers, was investigated by mutation of isoleucine at position 265 of the M subunit in Rhodobacter sphaeroides. Isoleucine was changed to threonine, serine, and valine, yielding mutants M265IT, M265IS, and M265IV, respectively. All three mutants, with smaller residues replacing isoleucine, exhibited decreased binding affinities of the Q(A) site for various quinone analogues, consistent with an enlargement or loosening of the headgroup binding domain and a decrease in the van der Waals contact for small quinones. In all other respects, M265IV was like the wild type, but the polar mutants, M265IT and M265IS, had unexpectedly dramatic decreases in the redox midpoint potential of Q(A), resulting in faster rates of P(+)Q(A)(-) charge recombination. For both anthraquinone and native ubiquinone, the in situ E(m) of Q(A) was estimated to be approximately 100 and 85 mV lower in M265IT and M265IS, respectively. The effect on E(m)(Q(A)) indicates destabilization of the semiquinone or stabilization of the quinone. This is suggested to arise from either (i) electrostatic interaction between the partial charges or dipole of the residue hydroxyl group and the charge distribution of quinone and semiquinone states with particular influence near the C4 carbonyl group or (ii) from hydrogen bonding interactions between the hydroxyl oxygen and the N(delta)H of histidine M219, causing a weakening of the hydrogen bond to the C4 carbonyl. The rate of the first electron transfer (k(AB)(()(1)())) in the polar mutants was the same as in the wild type at low pH but decelerated at higher pH with altered pH dependence. The rate of the second electron transfer (k(AB)(()(2)())) was 3-4-fold greater than in the wild type over the whole pH range from 4 to 11, consistent with a larger driving force for electron transfer derived from the lower E(m) of Q(A).

Preferential binding of equine ferricytochrome c to the bacterial photosynthetic reaction center from Rhodobacter sphaeroides.

Redox titration of horse heart cytochrome c (cyt c), in the presence of varying concentrations of detergent-solubilized photosynthetic reaction center (RC) from Rhodobacter sphaeroides, revealed an RC concentration-dependent decrease in the measured cyt c midpoint potential that is indicative of a 3.6 +/- 0.2-fold stronger binding affinity of oxidized cytochrome to a single binding site. This effect was correlated with preferential binding in the functional complex by redox titration of the fraction of RCs exhibiting microsecond, first-order, special pair reduction by cytochrome. A binding affinity ratio of 3.1 +/- 0.4 was determined by this second technique, confirming the result. Redox titration of flash-induced intracomplex electron transfer also showed the association in the electron transfer-active complex to be strong, with a dissociation constant of 0.17 +/- 0.03 microM. The tight binding is associated with a slow off-rate which, in the case of the oxidized form, can influence the kinetics of P(+) reduction. The pitfalls of the common use of xenon flashlamps to photoexcite fast electron-transfer reactions are discussed with relation to the first electron transfer from primary to secondary RC quinone acceptors. The results shed some light on the diversity of kinetic behavior reported for the cytochrome to RC electron-transfer reaction.

DCCD inhibits the reactions of the iron-sulfur protein in Rhodobacter sphaeroides chromatophores.

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. To understand the possible role of DCCD in inhibiting the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores. DCCD has two distinct effects on phase III of the electrochromic bandshift of carotenoids reflecting the electrogenic reactions of the bc(1) complex. At low concentrations, DCCD increases the magnitude of the electrogenic process because of a decrease in the permeability of the membrane, probably through inhibition of F(o)F(1). At higher concentrations (>150 microM), DCCD slows the development of phase III of the electrochromic shift from about 3 ms in control preparations to about 23 ms at 1.2 mM DCCD, without significantly changing the amplitude. DCCD treatment of chromatophores also slows down the kinetics of flash-induced reduction of both cytochromes b and c, from 1.5-2 ms in control preparations to 8-10 ms at 0.8 mM DCCD. Parallel slowing of the reduction of both cytochromes indicates that DCCD treatment modifies the reaction of QH(2) oxidation at the Q(o) site. Despite the similarity in the kinetics of both cytochromes, the onset of cytochrome c re-reduction is delayed 1-2 ms in comparison to cytochrome b reduction, indicating that DCCD inhibits the delivery of electrons from quinol to heme c(1). We conclude that DCCD treatment of chromatophores leads to modification of the rate of Q(o)H(2) oxidation by the iron-sulfur protein (ISP) as well as the donation of electrons from ISP to c(1), and we discuss the results in the context of the movement of ISP between the Q(o) site and cytochrome c(1).

Aspartate-187 of cytochrome b is not needed for DCCD inhibition of ubiquinol: cytochrome c oxidoreductase in Rhodobacter sphaeroides chromatophores.

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit steady-state proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. In chromatophores of the purple bacterium Rhodobacter sphaeroides, this has been associated with the specific labeling of a surface-exposed aspartate-187 of the cytochrome b subunit of the bc(1) complex [Wang et al. (1998) Arch. Biochem. Biophys. 352, 193-198]. To explore the possible role of this amino acid residue in the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and the electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores from wild type (WT) and mutant cells, in which aspartate-187 of cytochrome b (Asp(B187)) has been changed to asparagine (mutant B187 DN). The kinetics and amplitude of phase III of the electrochromic shift of carotenoids, reflecting electrogenic reactions in the bc(1) complex, and of the redox changes of cytochromes and reaction center, were similar (+/- 15%) in both WT and B187DN chromatophores. DCCD effectively inhibited phase III of the carotenoid bandshift in both B187DN and WT chromatophores. The dependence of the kinetics and amplitude of phase III of the electrochromic shift on DCCD concentration was identical in WT and B187DN chromatophores, indicating that covalent modification of Asp(B187) is not specifically responsible for the effect of DCCD-induced effects of cytochrome bc(1) complex. Furthermore, no evidence for differential inhibition of electrogenesis and electron transport was found in either strain. We conclude that Asp(B187) plays no crucial role in the protonogenic reactions of bc(1) complex, since its replacement by asparagine does not lead to any significant effects on either the electrogenic reactions of bc(1) complex, as revealed by phase III of the electrochromic shift of carotenoids, or sensitivity of turnover to DCCD.

Antisense inhibition of IGF receptor expression in HaCaT keratinocytes: a model for antisense strategies in keratinocytes.

Antisense strategies targeting skin conditions are attractive in concept, with a number of possible pathologic conditions, such a psoriasis, apparently suitable for such an approach. Because in vitro screening of candidate sequences is usually desirable, we have attempted to use a range of new generation cationic lipids to produce significant antisense oligodeoxynucleotide (ODN) uptake in an immortalized keratinocyte cell line (HaCaT). A large number of commercially available lipids were screened for the ability to induce nuclear ODN localization: Tfx-50, Tfx-20, Tfx-10, Superfect, Cytofectin GSV, Perfect lipids 1-8, Lipofectin, and Lipofectamine. All lipids were used at a range of concentrations (1-20 microg/ml) and with a range of ODN concentrations (1-1000) nM). Of all lipids used, only Cytofectin GSV and Superfect produced significant (>30% of cells) levels of nuclear positive cells, with Superfect also producing significant toxicity at the effective concentration used. Only two treatments produced a significant reduction in target mRNA: insulin-like growth factor-1 receptor (IGF-1R)-ODN 64 complexed with Cytofectin GSV (27.1% +/- 3.5% of IGF-1R mRNA in untreated cells,p < 0.01) and ODN 64 complexed with 10 microg/ml Lipofectin (62.2% +/- 3.4% of IGF-1R mRNA in untreated cells, p < 0.05). Only one treatment, ODN 64 complexed with Cytofectin GSV, produced a reduction in cell growth and survival as assessed by amido black assay. These results demonstrate that in HaCaT keratinocytes, Cytofectin GSV alone of all commercially available cationic lipids was effective in delivering antisense ODN into cell nuclei such that a profound antisense effect could be demonstrated.