Combined non-pharmacological interventions for newborn pain relief in two degrees of pain procedures: A randomized clinical trial.

BACKGROUND: Non-pharmacological interventions are effective neonatal pain reduction strategies. We aimed to study the effects of non-nutritive sucking (NNS) and swaddling on infants' behavioural and physiological parameters during shallow or deep heel stick procedures. METHOD: In this prospective, multi-centred, randomized controlled clinical trial, we enrolled 671 newborns. The infants undergoing shallow or deep heel stick procedures were randomized into four groups: oral sucrose (routine care, group S), oral sucrose combined with NNS (group NS), oral sucrose combined with swaddling (group SS) and oral sucrose combined with NNS and swaddling (group NSS). The behavioural responses were evaluated by the Revised Neonatal Facial Coding System and the physiological signals were monitored by electrocardiogram monitors. RESULTS: A significant synergistic analgesic effect was observed between the NS and SS groups in both the shallow (F = 5.952, p = 0.015) and deep heel stick (F = 7.452, p = 0.007) procedure. NSS group exhibited the lowest pain score. For the deep heel stick procedure, the NS group had a significantly lower increase in heart rate (HR)% and decrease in SPO2 % than the S group (F = 17.540, p = 0.000, F = 10.472, p = 0.001), while this difference was not observed in the shallow heel stick procedure. No difference was found between the S and SS groups, in terms of different physiological parameters. CONCLUSION: Non-nutritive sucking and swaddling had synergistic effects on pain relief when used with oral sucrose. For the deep heel stick procedure, oral sucrose combined with NNS and swaddling provided the best pain relief effect. For the shallow heel stick procedure, addition of NNS and swaddling did not improve the effects.

The N-terminus of gp130 is critical for the formation of the high-affinity interleukin-6 receptor complex.

Interleukin-6 (IL-6) mediates its activity through binding to two cell-surface receptors. The high-affinity human IL-6 receptor complex consists of two transmembrane anchored subunits: a ligand-specific, low-affinity IL-6 receptor and the high-affinity converter and signal transducing, gp130. Previously, using recombinant forms of human IL-6 and the extracellular ('soluble') domains of the IL-6 receptor (sIL-6R) and gp130 (sgp130), we have shown that the high-affinity IL-6R complex is hexameric, consisting of two molecules each of IL-6, sIL-6R and sgp130 (Ward et al., 1994, J. Biol. Chem. 269: 23286-23289). This paper investigates the role of the N-terminal region of gp130 in the formation of the high-affinity IL-6R complex. Using recombinant sgp130 produced with a FLAG octapeptide epitope (DYKDDDDK) at the N-terminus (sgp130-FLAG), we demonstrate, using biosensor analysis and size-exclusion chromatography, that modification of the N-terminus of sgp130 interferes with the in vitro in solution formation of the stable hexameric IL-6 receptor complex. Rather, sgp130-FLAG interacts with IL-6 and sIL-6R with a much lower affinity and forms a stable lower-order ternary complex. However, this lower-order complex is inconsistent with the solution molecular weight of a trimeric complex, as measured by size-exclusion chromatography. In contrast, N-terminal modification of the sgp130 with the FLAG epitope did not interfere with the binding of leukemia inhibitory factor or oncostatin-M (other cytokines that signal through gp130) to sgp130. These data support our model of the hexameric IL-6 receptor complex, which is biased towards the association of two IL-6.IL-6R.gp130 trimers, and postulates the critical involvement of the N-terminal Ig-like domain of gp130 in tethering the two trimers to form the stable hexamer (Simpson et al., 1997, Prot. Sci. 6: 929-955).

The human A33 antigen is a transmembrane glycoprotein and a novel member of the immunoglobulin superfamily.

The mAb A33 detects a membrane antigen that is expressed in normal human colonic and small bowel epithelium and > 95% of human colon cancers. It is absent from most other human tissues and tumor types. The murine A33 mAb has been shown to target colon cancer in clinical trials, and the therapeutic potential of a humanized antibody is currently being evaluated. Using detergent extracts of the human colon carcinoma cell lines LIM1215 and SW1222, in which the antigen is highly expressed, the molecule was purified, yielding a 43-kDa protein. The N-terminal sequence was determined and further internal peptide sequence obtained following enzymatic cleavage. Degenerate primers were used in PCRs to produce a probe to screen a LIM1215 cDNA library, yielding clones that enabled us to deduce the complete amino acid sequence of the A33 antigen and express the protein. The available data bases have been searched and reveal no overall sequence similarities with known proteins. Based on a hydrophilicity plot, the A33 protein has three distinct structural domains: an extracellular region of 213 amino acids (which, by sequence alignment of conserved residues, contains two putative immunoglobulin-like domains), a single hydrophobic transmembrane domain, and a highly polar intracellular tail containing four consecutive cysteine residues. These data indicate that the A33 antigen is a novel cell surface receptor or cell adhesion molecule in the immunoglobulin superfamily.

Purification and characterization of a novel restricted antigen expressed by normal and transformed human colonic epithelium.

A cell surface antigen that is expressed by normal and 95% of transformed colonic epithelium and is recognized by the monoclonal antibody A33 (Welt, S., Divgi, C. R., Real, F. X., Yeh, S. D., Garin-Chesa, P., Finstad, C. L., Sakamoto, J., Cohen, A., Sigurdson, E. R., Kemeny, N., Carswell, E. A., Oettgen, H. F., and Old, L. J. (1990) J. Clin. Oncol. 8, 1894-1906) has been purified to homogeneity from the human colonic carcinoma cell line LIM1215. The A33 protein was purified from Triton X-114 extracts of LIM1215 cells under nondenaturing conditions. These extracts were applied sequentially to Green-Sepharose HE-4BD, Mono-Q HR 10/10, Superose 12 HR 10/30, and micropreparative Brownlee Aquapore RP 300. The purification was monitored by biosensor analysis using surface plasmon resonance detection with a F(ab')2 fragment of the humanized A33 monoclonal antibody immobilized on the sensor surface and Western blot analysis following SDS-polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions using humanized A33 monoclonal antibody. The purified A33 antigen has a Mr on SDS-PAGE of 43,000 under nonreducing conditions. By contrast, the purified protein displayed a Mr of approximately 180,000 under native conditions on both size exclusion chromatography and native PAGE, possibly due to the formation of a homotetramer. N-terminal amino acid sequence analysis of the purified protein identified 34 amino acid residues of a unique sequence: ISVETPQDVLRASQGKSVTLPXTYHTSXXXREGLIQWD. A polyclonal antibody was raised against a synthetic peptide corresponding to residues 2-20 of this sequence. The antipeptide serum recognized the purified protein using Western blot analysis under both nonreducing (Mr 43,000) and reducing (Mr 49,000) conditions.

Complete nucleotide sequence, expression, and chromosomal localisation of human mixed-lineage kinase 2.

Protein kinases play pivotal roles in the control of many cellular processes. In a search for protein kinases expressed in human epithelial tumour cells, we discovered two members of a novel protein kinase family [Dorow, D. S., Devereux, L., Dietzsch, E. & de Kretser, T. A. (1993) Eur. J. Biochem. 213, 701-710]. Due to the unique mixture of structural domains within their amino acid sequences, we named the family mixed-lineage kinases (MLK). We initially isolated clones encoding partial cDNAs of MLK1 and 2 from a human colonic cDNA library. The MLK2 cDNA was subsequently used to screen a human brain cDNA library and we have now cloned and sequenced a 3454-bp cDNA encoding the full-length MLK2 protein. The predicted MLK2 polypeptide has 954 amino acids and contains a src homology 3 (SH3) domain, a kinase catalytic domain, a double leucine zipper and basic domain, and a large C-terminal domain. The 22-amino-acid N-terminal region has four glutamic acid residues immediately following the initiator methionine. Beginning at amino acid 23, the 55-amino-acid SH3 domain contains a 5-amino-acid insert in a position corresponding to inserts of 6 and 15 residues in the SH3 domains of n-src and the phosphatidylinositol 3'-kinase. Adjacent to the SH3 domain is a kinase catalytic domain with conserved motifs associated with both serine/threonine and tyrosine specificity. Beginning nine residues C-terminal to the catalytic domain, there are two leucine/isoleucine zippers separated by a 13-amino-acid spacer sequence and followed by a stretch of basic residues. The polybasic sequence contains a motif that is similar to nuclear localisation signals from several proteins. The C-terminal domain is composed of 491 amino acids of which 17% are serine or threonine and 16% are proline. This domain also has a biased ratio of basic to acidic amino acids with a calculated pI of 9.38. When used as a probe to examine mRNA expression in human tissues, a MLK2 cDNA hybridised to a species of 3.8 kb that was expressed at highest levels in RNA from brain and skeletal muscle. The 3454-bp cDNA was also used for fluorescence in situ hybridisation to localise the MLK2 gene to human chromosome 19 q13.2.

C-terminal extension of truncated recombinant proteins in Escherichia coli with a 10Sa RNA decapeptide.

When murine interleukin-6 is overexpressed in Escherichia coli, a small population of molecules exhibits a novel C-terminal modification. Peptide mapping, electrospray ionization-mass spectrometry, and automated N- and C-terminal sequencing identified a peptide ("tag" peptide), -Ala-Ala-Asn-Asp-Glu-Asn-Tyr-Ala-Leu-Ala-Ala-COOH, encoded by a small metabolically stable RNA of E. coli (10Sa RNA) attached to truncated C termini of the recombinant protein. A mutant strain of E. coli in which the chromosomal 10Sa RNA gene (ssrA) is disrupted does not produce this C-terminal modification, confirming that the tag peptide originates from the ssrA gene.

Strong conservation of the expression of cystatin C gene in choroid plexus.

The expression of the cystatin C gene was studied by Northern analysis of RNA isolated from the choroid plexus, other brain tissues, and liver from 11 mammalian and 4 avian species. The probe used for hybridization was cystatin C cDNA isolated previously from a rat choroid plexus cDNA library. Strong conservation of the expression of the cystatin C gene in choroid plexus was suggested by the observation of substantial levels of cystatin C mRNA in choroid plexus RNA from all mammalian and avian species studied. In contrast, levels of cystatin C mRNA in total liver RNA varied widely for mammalian as well as for avian species. It was concluded that the synthesis of cystatin C in choroid plexus has probably been conserved since the stage of the stem reptiles, the common ancestors of mammals and birds. The cystatin C gene was also found to be expressed early in ontogeny, as indicated by the observation of similar cystatin C mRNA levels in choroid plexus RNA from newly hatched and adult chickens.

Species specificity and developmental patterns of expression of the beta amyloid precursor protein (APP) gene in brain, liver and choroid plexus in birds.

1. Human APP cDNA hybridized to a 3.5 kb mRNA in liver and brain RNA from chickens, pigeons, quail and ducks as well as in RNA from choroid plexus of chicken and quail. In contrast to all other species hitherto examined a 1.6 kb mRNA hybridizing to APP cDNA was found in abundant amounts in RNA from chicken and quail livers. 2. In the chicken, before hatching, the levels of APP mRNA in total RNA from liver and choroid plexus were higher than those in RNA from liver and choroid plexus of adults. However, RNA from the rest of the brain of chicken embryos contained less APP mRNA than RNA from brain of adults. 3. In the chicken, between 10 and 40 days after hatching, APP mRNA levels in RNA from liver were higher than adult levels, APP mRNA levels in RNA from choroid plexus were similar to adult levels and APP mRNA levels in RNA from the rest of brain were below the adult levels.

Cerebral expression of transthyretin: evolution, ontogeny and function.

This paper reviews studies on the synthesis and secretion of the thyroid hormone-binding protein, transthyretin by the choroid plexus. The secretion of transthyretin by the choroid plexus into the cerebrospinal fluid may have an important function in the transport of thyroxine from the blood to the brain. The transthyretin gene is expressed in the choroid plexus of most vertebrates and synthesis of this protein may have evolved in the brain before the liver.

The distribution of cerebral expression of the transferrin gene is species specific.

Various plasma proteins, for example, transferrin, are synthesized not only in the liver, but also in the brain. The proportion of transferrin mRNA in total RNA from different regions of brains from various mammalian species was studied by Northern blot analysis. Absolute amounts of transferrin mRNA were determined in brain, choroid plexus, and liver from rats, sheep, and pigs by hybridization in solution followed by ribonuclease protection assay. Corrections for differences in yields of RNA were made using internal RNA standards. Large proportions of transferrin mRNA in total RNA and high absolute levels of transferrin mRNA in choroid plexus were found only in rats. Small proportions of transferrin mRNA were observed in RNA from choroid plexus from mice, dogs, and rabbits, while no transferrin mRNA at all was detected in choroid plexus from humans, sheep, pigs, cows, and guinea pigs. In further analysis of sheep and pigs, various amounts of transferrin mRNA were found in many parts of the brain, in contrast to the absence of transferrin mRNA from choroid plexus. In conclusion, a striking species specificity was observed for the pattern of cerebral expression of the transferrin gene.

Transthyretin (prealbumin) gene expression in choroid plexus is strongly conserved during evolution of vertebrates.

1. The major protein synthesized and secreted by the choroid plexus from mammals, birds, reptiles and probably amphibians is similar in subunit structure to transthyretin. 2. In mammals and birds the proportion of transthyretin mRNA is much higher in choroid plexus RNA than in liver RNA. No transthyretin mRNA is found in brain outside the choroid plexus. 3. Transthyretin-like protein, such as that secreted by the choroid plexus, was not detected in amphibian serum and was present in very low levels in reptile serum. 4. It is proposed that transthyretin synthesis and secretion arose earlier in evolution in the choroid plexus than in the liver.

Ontogenesis of transthyretin gene expression in chicken choroid plexus and liver.

1. Chicken liver transthyretin cDNA hybridizes strongly with choroid plexus transthyretin mRNA from chickens, pigeons, quails and ducks. 2. In the chicken at hatching the choroid plexus has reached 70%, total brain 30%, and liver 5.8% of their organ masses in adults. 3. The proportion of transthyretin mRNA in total RNA is 0.45-times the adult value in the choroid plexus of the chicken at hatching. 4. In the liver at hatching, the proportion of transthyretin mRNA in total RNA is 1.1-times the value in adult chickens. 5. The pattern of maturation of transthyretin gene expression in chicken liver is comparable to that in precocial, but differs from that in altricial mammals.

Expression of the genes for transthyretin, cystatin C and beta A4 amyloid precursor protein in sheep choroid plexus during development.

The levels of mRNA for transthyretin, cystatin C, and beta A4 amyloid precursor protein were measured in the choroid plexus of sheep embryos during different stages of development, using specific cDNA probes and Northern blot analysis. The 3 different mRNAs were detectable in the brain of very young embryos with a crown-rump length of 1 cm, corresponding to only a few days of gestation. The choroid plexus increased in weight very rapidly in the first half of gestation and much more slowly in the second half. The level of transthyretin mRNA in choroid plexus increased during the first half of gestation and stayed constant thereafter until birth, at a level of about 70% of that in choroid plexus of adult sheep. The proportion of mRNA for the proteinase inhibitor cystatin C in total RNA from choroid plexus increased throughout gestation to adult levels at birth. The concentration of the mRNA for beta A4 amyloid precursor protein in choroid plexus early in development was already as high as in adults and remained at this level throughout gestation. Messenger RNA for cystatin C or mRNA for beta A4 amyloid precursor protein was not detected in adult sheep liver.

Effect of acute inflammation on rat apolipoprotein mRNA levels.

Hybridization studies using specific cDNA have been used to determine the mRNA levels for rat apolipoproteins AI, AII, AIV, and E in extracts of rat liver and intestine. The ratios of intestinal mRNA/liver mRNA for apolipoprotein AI (apo AI), apo AIV, and apo E were 1.3, 1.7, and 0.1, respectively. Apo AII mRNA was detected in the liver but not in the intestine. The mRNA levels for apo AII and apo AIV in rat liver decreased during inflammation to minimums of 40% and 25% of normal, respectively. The mRNA levels for apo AIV in the intestine, apo E in the liver and for apo AI in both the liver and intestine did not change significantly during inflammation. The time course for the decrease in the hepatic mRNA levels for apo AIV was similar to those previously observed for the negative acute-phase proteins albumin and transthyretin. The serum levels for apo AIV were not affected by inflammation.

Isolation and characterization of genes for blood proteins.

The expression vector lambda gt11Amp3 has been used to construct a cDNA library from rat liver polyadenylated RNA. Clones expressing antigenic determinants for rat albumin, transferrin, transthyretin, apolipoprotein E and apolipoprotein AII have been identified. Albumin clones containing cDNA inserts ranging from 0.9 kb to 1.9 kb were further identified by restriction mapping and nucleic acid sequencing. The largest insert contained the entire coding sequence for albumin. Characterization of the expressed proteins by acrylamide gel electrophoresis followed by immunological detection indicated that the proteins were produced as hybrids linked to the bacterial beta-galactosidase. A cDNA library for human liver polyadenylated RNA has also been constructed. Clones expressing antigenic determinants for human serum albumin, transferrin and apolipoproteins AI, AII, AIV and E have been isolated and their identity established by nucleotide sequencing and restriction mapping. Both rat and human serum protein cDNA clones are currently being used to study the tissue specific expression of serum proteins and for the isolation and characterization of the corresponding genes.

Levels of messenger ribonucleic acids for plasma proteins in rat liver during acute experimental inflammation.

The levels of mRNA for plasma proteins and for metallothionein in rat liver during the acute-phase response were studied by hybridization to specific cDNA probes. The mRNA for alpha 2-macroglobulin, the beta-chain of fibrinogen, alpha 1-acid glycoprotein (so-called acute-phase reactants) reached a maximum level between 18 and 36 h after inducing an acute inflammation. The level of mRNA for metallothionein-I peaked earlier, after 12 h. The mRNA for transferrin showed a delayed increase with a broad maximum for its relative level after 36-60 h. The mRNA levels for albumin and alpha 2u-globulin (so-called negative acute-phase reactants) decreased, reaching a minimum of 25% of the normal level after 36 h (albumin) and after 72 h (alpha 2u-globulin). The ratios of the rates of incorporation of leucine into the proteins over the levels of their mRNA in liver changed only a little, indicating that the rates of synthesis of plasma proteins in the liver are regulated at the mRNA level during the acute-phase response to inflammation.

High prealbumin and transferrin mRNA levels in the choroid plexus of rat brain.

Expression of plasma protein genes in various parts of the rat brain was studied by hybridizing radioactive cDNA to RNA in cytoplasmic extracts. No mRNA could be detected in brain for the beta subunit of fibrinogen, major acute phase alpha 1-protein, alpha 1-acid glycoprotein and albumin. However, per g tissue, the choroid plexus contained at least 100 times larger amounts of prealbumin mRNA than the liver and about the same amount of transferrin mRNA as liver. No prealbumin mRNA was found in other areas of the brain. The results obtained suggest very active synthesis of prealbumin in choroid plexus, which would be an important link in the transport of thyroid hormones from the blood to the brain via the cerebrospinal fluid.