Gene delivery by cationic lipids: in and out of an endosome.

Cationic lipids are exploited as vectors ('lipoplexes') for delivering nucleic acids, including genes, into cells for both therapeutic and cell biological purposes. However, to meet therapeutic requirements, their efficacy needs major improvement, and better defining the mechanism of entry in relation to eventual transfection efficiency could be part of such a strategy. Endocytosis is the major pathway of entry, but the relative contribution of distinct endocytic pathways, including clathrin- and caveolae-mediated endocytosis and/or macropinocytosis is as yet poorly defined. Escape of DNA/RNA from endosomal compartments is thought to represent a major obstacle. Evidence is accumulating that non-lamellar phase changes of the lipoplexes, facilitated by intracellular lipids, which allow DNA to dissociate from the vector and destabilize endosomal membranes, are instrumental in plasmid translocation into the cytosol, a prerequisite for nuclear delivery. To further clarify molecular mechanisms and to appreciate and overcome intracellular hurdles in lipoplex-mediated gene delivery, quantification of distinct steps in overall transfection and proper model systems are required.

Supplemental lactoferrin improves health and growth of Holstein calves during the preweaning phase.

Lactoferrin is a milk protein that exhibits broad-spectrum antimicrobial properties. Previous studies indicated that supplemental lactoferrin may alter the microbial populations in the gut of nonruminants and increase preweaning weight gains in calves. In the present study, 40 Holstein calves were used to examine the effects of supplemental lactoferrin (0, 1, 2, or 3 g/d) on health, growth, and feed intake from 3 d of age to 2 wk postweaning. Lactoferrin was mixed and fed with a nonmedicated milk replacer. Calves were housed in individual pens and offered a textured, nonmedicated starter and water for ad libitum consumption. Body weight and heart girth were measuredweekly. Intakes of milk replacer and starter were determined daily. Fecal consistency was monitored three times per week. Calves were weaned when they met certain criteria based on body weight gain and starter intake. Preweaning fecal score responded quadratically, with the group fed 1 g/d of lactoferrin having the lowest score. Overall and preweaning number of days medicated responded in the same manner as fecal score. Preweaning average daily gain and gain-to-feed ratio increased linearly with lactoferrin supplementation, whereas postweaning gain-to-feed ratio decreased linearly with lactoferrin. Overall average daily heart girth gain increased linearly with lactoferrin. Body weight, weaning age, and dry matter intake were not different among treatments. Based on the observed improved gain-to-feed ratios, increased average daily gains, improved fecal scores, and reduced morbidity in preweaned calves, it appears that lactoferrin may be a beneficial supplement in the diets of neonatal calves prior to weaning.

Lactoferrin supplementation to dairy calves.

Twenty-one Holstein calves (12 bulls, 9 heifers) were used to evaluate the effects of supplemental lactoferrin (0, 1, and 10 g/d) added to colostrum, milk, and milk replacer in a 56-d study. Calves fed lactoferrin (LF) weighed more during wk 2, 3, 4, 5, and 6 than control calves. Calves fed LF had increased preweaning daily weight gains and heart girth gains compared with calves not fed lactoferrin. Calves fed 1 g/d LF had a greater preweaning average daily gain than calves fed 10 g/d of lactoferrin. Hematocrit and serum Fe were unaffected by treatments. Calves were weaned when they had consumed 0.7 kg of calf starter grain for 2 consecutive d and were at least 28 d of age. All calves were weaned by 35 d of age, regardless of starter intake. Calves fed LF consumed more calf starter grain during the preweaning period and met weaning criteria at a younger age. This study indicates that supplementing calf diets with LF is advantageous to calf performance.

cDNA cloning and gene expression of the type 1 bovine interleukin-1 receptor.

Regulation of interleukin-1 (IL-1) mediated biological responses is complicated by the multiple ligands and receptors of the IL-1 family. Most studies of IL-1 receptors have used human or rodent cells. Here, we report that the coding region of the bovine type 1 interleukin-1 receptor (type 1 IL-1R) cDNA extends 1719 bp in length. Northern analysis of specific bovine cell and tissue RNA demonstrated a 4.5 kb transcript. Overall, the bovine type 1 IL-1R coding region exhibits approximately 81 and 76% similarity with the human type 1 IL-1R at the nucleotide and amino acid level, respectively, and somewhat less similarity with the mouse and rat sequences. Type 1 IL-1R transcripts were confirmed by RT-PCR in several bovine cell types, including peripheral blood mononuclear cells (PBMCs), neutrophils (PMNs), and fibroblast, peritoneal macrophage, and arterial endothelial cell lines. It is expected that molecular clones for the bovine type 1 and 2 IL-1 receptors will provide us with the tools needed to decipher species-and cell-specific regulation of IL-1 action in the bovine.

Regulation of bovine interleukin-1 receptors.

Interleukin-1 is a key player in inflammation and the immune response. The interleukin-1 family consists of three ligands (IL-1 alpha, IL-1 beta, and the IL-1 receptor antagonist) and two receptors (IL-1RI and IL-1RII). Previous studies suggest a dynamic relationship among these receptors and ligands that regulates the magnitude and extent of IL-1 mediated activities. Our laboratory has cloned and sequenced the bovine type I and II interleukin-1 receptors, and has begun to investigate their regulation in bovine leukocytes in vitro and in vivo. IL-1RI and IL-1RII mRNA levels were upregulated in vitro by various mediators, including dexamethasone, rBoIL-4, rBoGM-CSF, and rHuTNF alpha. Conversely, IL-1RI mRNA levels were down-regulated by IFN-gamma. An in vivo study indicated that IL-1RII mRNA levels increased earlier than IL-1RI mRNA levels in dexamethasone-treated cattle. These findings suggest that early upregulation of IL-1RII, which is a decoy receptor, may be part of the anti-inflammatory action of glucocorticoids. Our investigations suggest that anti-inflammatory agents increase expression of the biologically inactive IL-1RII, as compared with the biologically active IL-1RI, in bovine leukocytes.

Active immunization of Japanese quail hens with a recombinant chicken inhibin fusion protein enhances production performance.

The effects of active immunization against inhibin on production performance in female Japanese quail (Coturnix coturnix japonica) were assessed in two separate trials using an MBP-cINA521 fusion protein as an immunogen. The fusion protein, MBP-cINA521, consisted of the bacterial maltose binding protein (MBP) and a truncated form of the mature alpha-subunit of chicken inhibin (cINA521). MBP-cINA1521 was constructed by: 1) excising a 521-bp PstI fragment from a chicken inhibin alpha-subunit cDNA (cINA6; gift of P. A. Johnson), 2) cloning this fragment, which encodes all but the first 11 amino acid residues of the mature alpha-subunit, into the pMal-c2 vector of the MBP fusion expression system, and 3) expressing the fusion protein (MBP-cINA521) from the Escherichia coli and purifying it using affinity chromatography. In each trial, quail were randomly and equally assigned to one of two injection treatments as follows: 1) MBP-cINA521 in Freund's adjuvant, or 2) Freund's adjuvant (vehicular controls; CON). All immunizations were given subcutaneously and Freund's complete and incomplete adjuvant were used for primary and booster injections, respectively. In Trial 1, birds were given a primary challenge of 0.2 mg MBP-cINA521 per bird at 25 d of age, followed by booster immunizations (0.1 mg MBP-cINA521 per bird) at 33, 40, 47, 54 and 61 d of age and every 35 d thereafter. The CON birds received vehicular immunizations at the same time intervals. In Trial 2, birds treated with MBP-cINA521 received a primary challenge of 0.2 mg MBP-cINA521 per bird at 26 d of age, followed by booster immunizations (0.1 mg MBP-cINA521 per bird) using the same schedule as that used in Trial 1, with the exception that no boosters were given after 61 d of age. The CON birds received vehicular immunizations at the same time intervals. Collection of production performance data was initiated coincident with the laying of the first egg in each trial (i.e., beginning at 41 and 44 d of age for Trials 1 and 2, respectively) and continued for 30 1-wk periods of lay. Combined data from Trials 1 and 2 indicated that the mean +/- SE age at first egg lay was markedly decreased (P < 0.005) in MBP-cINA521-treated quail (53.4 +/- 0.9 d of age) when compared to the CON (57.6 +/- 1.3 d of age). Likewise, the mean +/- SE age at 50% egg production was reduced (P < 0.03) in quail immunized against inhibin (65.4 +/- 2.1 d of age) when compared to the CON (77.6 +/- 4.7 d of age). Total hen-day egg production was also higher (P < 0.05, Trial 1; P < 0.01, Trial 2) in MBP-cINA521-treated quail (88.7 +/- 1.4%, Trial 1; 90.1 +/- 1.2%, Trial 2) than in the CON birds (81.9 +/- 2.9%, Trial 1; 73.6 +/- 6.5%, Trial 2). Collectively, these findings provide evidence that inhibin immunoneutralization accelerated puberty and enhanced hen-day egg production during a 30-wk period of egg lay in Japanese quail.

Changes in bovine mammary-secretion composition during early involution following intramammary infusion of recombinant bovine cytokines.

This study was designed to determine whether intramammary infusion of recombinant bovine interleukin-1 beta or interleukin-2 had any adverse influence on bovine mammary function during the early non-lactating period. Mammary glands of eight Jersey cows were infused with either 10 micrograms of interleukin-1 beta or 1.0 mg of interleukin-2 following abrupt cessation of milking. Mammary secretions from each gland were collected from cows frequently during early involution and evaluated for changes in secretion composition. Percentage of milk fat and solids-not-fat during the experimental period was variable for all treatments. Percentage of protein increased throughout the study for all treatments, but mammary glands treated with interleukin-1 beta had higher protein percentages. All interleukin-treated mammary glands had lower concentrations of lactose than controls. Mammary secretions from interleukin-1 beta treated glands had higher concentrations of lactoferrin at 7 and 14 days of involution. Similarly, mammary secretions from interleukin-2 treated glands had higher concentrations of serum albumin on day 3 of involution and throughout the remainder of the study. Concentration of citrate and the citrate: lactoferrin molar ratio generally decreased throughout the study for all treatment groups. Concentration of alpha-lactalbumin in mammary secretions decreased throughout the study and was not different between treatment groups. Though significant changes in mammary secretion composition due to the presence of cytokines were observed in this study, resulting in an apparent acceleration of involution, there was no indication that these changes adversely affected normal bovine mammary function during the early non-lactating period.

[Congenital defects as a cause of infant mortality in Zielona Góra province in 1987-1992]. / Wady wrodzone jako przyczyna umieralnosci niemowlat w województwie zielonogórskim w latach 1987-1992.

A total of 1278 children with congenital defects born in Zielona Góra Province between 1987 and 1992 were studied. Of this group, 315 children (24,6%) died. Congenital defects were responsible for 38.1% of these deaths and were the main cause of late infant mortality. Congenital heart defects were the most frequent cause of death. Two hundred and ten (66%) of all of the deaths occurred in the neonatal period. Mortality from congenital defects among low birth weight neonates was twice the overall rate.

[The occurrence of congenital abnormalities in children under 2 years of age in Zielona Gora province between 1987-1992]. / Wystepowanie wad wrodzonych u dzieci do 2 roku zycia w województwie zielonogórskim w latach 1987-1992.

The analysis encompassed 1278 children under 2 years of age with congenital abnormalities from a group of 60, 815 live-born neonates in Zielona Góra province between 1987-1992. The population prevalence of congenital abnormalities was 2.10% on average. Cardiovascular system abnormalities were the most frequent, making up 40.53% of all detected abnormalities and 0.85% of the analyzed population. The successive positions were abnormalities of: the limbs (15.41% of all detected abnormalities and 0.31% of the analyzed population), genitourinary system (9.08% and 0.19% respectively), central nervous system (7.51% i 0.16%) and digestive system (6.57% and 0.14). There were no statistically significant differences between sexes, towns and villages, jobs of mothers and months of birth. Congenital abnormalities were twice as frequent (4.83%) in low-birth-weight neonates. Deaths caused by congenital abnormalities made 30.9% of total infantile mortality. 66% of children with congenital abnormalities (mainly heart defects) died in first month of life.

Proliferation of a bovine mammary epithelial cell line in the presence of bacterial virulence factors.

Effects of bacterial virulence factors on bovine mammary cell structure and function are not well defined. In this study, we evaluated the influence of specific bacterial virulence factors on proliferation of a bovine mammary epithelial cell line. The MAC-T cells were cultured in the presence of medium only, Staphylococcus aureus alpha-toxin, Staph. aureus beta-toxin, Escherichia coli endotoxin, Streptococcus uberis capsule, or hyaluronidase. Cells were cultured in the presence of virulence factors for 48 h at 37 degrees C. The MAC-T cell proliferation was inhibited by all concentrations of endotoxin and alpha-toxin and by most concentrations of hyaluronic acid capsule and hyaluronidase > 7.8 micrograms/ml. Staphylococcus aureus beta-toxin had no effect on MAC-T cell proliferation. Virulence factors produced by mastitis pathogens may influence mammary epithelial cell proliferation in vivo, which could be important during the periparturient period, when mammary tissue undergoes rapid differentiation and growth.

Characterization of lactoferrin binding to the MAC-T bovine mammary epithelial cell line using a biotin-avidin technique.

1. A non-radioisotopic method utilizing a biotin-avidin approach was used to characterize lactoferrin binding to the clonal MAC-T bovine mammary epithelial cell line. 2. Binding of lactoferrin to MAC-T cells and isolated membranes was specific and saturable. 3. Unlabeled lactoferrin competed for and displaced biotin-labeled lactoferrin from binding sites on mammary epithelial cells. In contrast, unlabeled transferrin did not compete. 4. Scatchard analysis of lactoferrin binding to MAC-T cell crude membranes was nonlinear, revealing two classes of binding sites with association constants (Ka) of 2.36 x 10(7) and 3.36 x 10(6) M-1. 5. Binding of lactoferrin to MAC-T cells may be associated with the initial events which result in decreased MAC-T cell proliferation.

Response of peripheral blood mononuclear cells to mitogenic stimulation in young calves.

Sixteen Holstein bull calves were used to evaluate effects of increasing blood BHBA, as a consequence of ruminal development, on response of peripheral blood mononuclear cells to mitogenic stimulation. Calves were blocked by date of birth and assigned to weaning at 35 or 49 d of the study. Calves began the study at 4 d of age. Milk replacer was fed at 10% of BW to d 7 prior to weaning; thereafter, milk replacer offered was reduced by 50%. Calf starter was offered from d 1. Blood was sampled at wk 0, 2, 4, 6, 8, and 10 at 4 h after the a.m. feeding. Blood glucose declined, and blood BHBA increased, as calf starter intake increased. Blood BHBA increased at weaning in each treatment group and exceeded 1 mM during the week of weaning. Responses of peripheral blood mononuclear cells to stimulation with concanavalin A, pokeweed mitogen, or phytohemagglutinin were unaffected by increasing blood BHBA and increased with age. Increased concentration of blood BHBA in young calves from increased starter intake did not impair immune response.

Effects of interleukin-1 and interleukin-2 on mammary gland leukocyte populations and histology during the early nonlactating period.

Eight Jersey cows free of intramammary infection (IMI) with major mastitis pathogens were intramammarily infused with interleukin (IL)-1, IL-2, or phosphate-buffered saline (PBS) after the last milking prior to drying off. Rectal temperatures were monitored hourly through 6 hours after infusion and at 24-hour intervals thereafter. Quarter secretions were collected 7 days prior to drying off, at drying off, and on days 1, 2, 3, 4, 7, and 14 of the nonlactating period. Cows were slaughtered and mammary tissues were collected at 7 (3 cows) and 14 (5 cows) days after treatment. The infusion of 10 micrograms of IL-1 produced an elevation in rectal temperature that was significantly higher than that of cows receiving IL-2 or PBS at 4, 5, and 6 hours after treatment, whereas cows receiving 1.0 mg of IL-2 exhibited no change in rectal temperature. Somatic cell counts (SCC) in mammary glands infused with IL-1 or IL-2 remained significantly elevated over PBS-infused control glands through day 4 after treatment. Neutrophils predominated in secretions of IL-1-treated glands, and macrophages and lymphocytes predominated in IL-2-treated glands. The mammary involution process was accelerated by IL-2, and tissues exhibited reductions in alveolar epithelial and luminal areas and increases in connective tissue stroma and degree of leukocytosis compared with IL-1 and control glands. Concentrations of plasma cells producing IgG1, IgG2, IgA, and IgM also were greatest following IL-2 infusion. Results suggest that IL-2 is superior to IL-1 in stimulating an increase in mammary mononuclear cell populations and in accelerating involution during the early nonlactating period.

Bovine lactoferrin in involuting mammary tissue.

Bovine lactoferrin in involuting mammary tissue was identified by immunohistochemistry and tissue explant culture. Immunoreactive lactoferrin was associated with mammary epithelial cells. Immunostaining for lactoferrin increased during involution, in contrast to declining immunostaining of epithelia for the milk-specific protein beta-lactoglobulin. Immunostaining for lactoferrin also was observed at the basal region of alveolar epithelia, perhaps in association with basement membrane components. Lactoferrin was preferentially synthesized in involuting mammary tissue compared with lactating tissue. Synthesis of lactoferrin in the involuting mammary gland occurs despite the apparent decline in synthesis of milk-specific proteins.

Proliferation of the MAC-T bovine mammary epithelial cell line in the presence of mammary secretion whey proteins.

Development of the MAC-T bovine mammary epithelial cell line by stable transfection with simian virus-40 large T-antigen should greatly assist study of possible intrinsic (local) and extrinsic (systemic) factors regulating bovine mammary epithelial cell development, differentiation, and function. This study evaluated the influence of mammary secretion whey proteins alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), lactoferrin (LF), transferrin (TF) and serum albumin (SA) on MAC-T cell proliferation in the absence and presence of 10% fetal bovine serum (FBS). Concentration of whey proteins in culture ranged from 0 to 625 micrograms/ml. MAC-T cell proliferation in the absence of FBS was significantly lower than in the presence of 10% FBS. Alpha-lactalbumin and LF significantly decreased MAC-T proliferation in both the absence and presence of 10% FBS. Transferrin significantly increased MAC-T cell proliferation only in the absence of FBS. There were no significant differences in MAC-T cell proliferation cultured in the presence of BLG or SA. These experiments illustrate the potential usefulness of MAC-T cells for the study of factors involved in mammary cell proliferation. Results identified ALA, LF and TF as possible intrinsic factors associated with regulation of mammary epithelial cell proliferation.

Purification of a nuclear protein (receptor binding factor-1) associated with the chromatin acceptor sites for the avian oviduct progesterone receptor.

The specific high affinity binding of the avian oviduct progesterone receptor (PR) to target cell nuclei and chromatin has been shown to involve DNA complexed with specific chromatin acceptor proteins. One of these chromatin acceptor proteins has been partially purified and found to be a small hydrophobic protein with a broad pI of 5.0-6.0 [Goldberger and Spelsberg (1988), Biochem. 27, 2103-2109]. Using western immunoblots with anti-RBF-1 polyclonal antibodies to monitor the purification, a 10 kD candidate acceptor protein, termed the Receptor Binding Factor-1 (RBF-1), has been purified to apparent homogeneity. RBF-1 has an amino acid composition consistent with a hydrophobic protein having an acidic pI and a unique N-terminal sequence. Two-dimensional polyacrylamide gel electrophoresis and high-performance capillary electrophoresis support the purity of a protein congruent to 10 kD in size, having an acidic pI, but with evidence of several differently charged isoforms. Phosphatase treatment provides evidence that charge heterogeneity may result from variable phosphorylation states. A role of this factor as a candidate "acceptor protein" in the chromatin acceptor sites for the avian oviduct PR is proposed.

Characterization of a purified chromatin acceptor protein (receptor binding factor 1) for the avian oviduct progesterone receptor.

The specific, high-affinity binding of the avian oviduct progesterone receptor (PR) with target-cell nuclei and chromatin has been shown to involve DNA complexed with specific chromatin acceptor proteins. One of these chromatin acceptor proteins has been partially purified and found to be a small hydrophobic protein with a broad pI of 5.0-6.0 [Goldberger, A., & Spelsberg, T. C., (1988) Biochemistry 27, 2103-2109]. This paper describes the final purification over 100,000-fold to apparent homogeneity of this candidate PR acceptor protein, termed the receptor binding factor 1 (RBF-1). When the avian genomic DNA is bound by RBF-1, saturable, high-affinity (KD approximately 2 x 10(-9) M) binding sites for PR are generated. RBF-1 has a unique, hydrophobic N-terminal sequence. The PR binding to the RBF-1-DNA complexes is shown to be dependent on an intact activated PR with which excess nonradiolabeled PR can compete. By use of a new, highly specific monoclonal antibody (mAb) to the RBF-1 with Western immunoblotting, RBF-1 was shown to be localized in the nucleus and to be tissue and species specific. Selective removal of the chromatin proteins containing RBF-1 results in the loss of the highest affinity class of PR binding sites. A second class of residual PR binding sites remains in the nucleoacidic protein (NAP), a complex of proteins more tightly bound to the DNA. This class of PR binding activity has been classified as the RBF-2. The RBF-1 is estimated to be 0.03% of the total chromatin protein with about 1.2 x 10(5) molecules/diploid cell.

Enzyme-linked immunosorbent assays of bovine lactoferrin and a 39-kilodalton protein found in mammary secretions during involution.

Enzyme-linked immunosorbent assays were developed for two proteins found in mammary secretions during the non-lactating period: lactoferrin and a 39-kdal protein. Minimum detectable concentrations were .12 ng/ml for lactoferrin and .0037 ng/ml for the 39-kdal protein. Standard curves were linear in the ranges of .4 to 100 ng/ml for lactoferrin and 2.4 to 30 ng/ml for the 39-kdal protein. The immunoassays were used to measure concentrations of the proteins in mammary secretions during involution. From the last day of milking to d 30 of involution, mean concentrations of lactoferrin and the 39-kdal protein increased from .82 +/- .35 to 78.5 +/- 12.43 mg/ml and from 11.8 +/- 3.8 to 84.4 +/- 21.1 micrograms/ml, respectively. The immunoassays described will be valuable for further studies on the synthesis and secretion of these proteins during mammary involution.

Purification and characterization of bovine lactoferrin from secretions of the involuting mammary gland: identification of multiple molecular weight forms.

1. Lactoferrin was isolated from bovine mammary secretions collected during the nonlactating period. 2. A method utilizing heparin-agarose affinity chromatography was more efficient for purifying lactoferrin than a method including gel filtration, ion exchange chromatography and a second gel filtration. 3. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the purified lactoferrin was composed of two protein bands of apparent mol. wt. of 83,000 and 87,000. 4. Digestion with endoglycosidase H resolved the lactoferrin into two lower mol. wt. bands of 78,000 and 81,000. 5. The biochemical differences between the forms of lactoferrin are not exclusively due to differences in endoglycosidase H-sensitive oligosaccharide composition.