Yoshihiro Fujiwara

Position‐independent and high‐level expression of human α‐lactalbumin in the milk of transgenic rats carrying a 210‐kb YAC DNA

The level of expression of transgenes in transgenic animals varies among lines, and is often much lower than that of endogenous genes (position effects). In order to surmount position effects and establish a more efficient production system of transgenic animals producing pharmaceutical proteins in their milk, transgenic rats carrying 210‐kb YAC DNA containing the human α‐lactalbumin gene were produced. Three transgenic lines transmitted the transgene to the next generation. They had one copy of the α‐lactalbumin gene and secreted human α‐lactalbumin in their milk at concentrations of 2.0–4.3 mg/ml. No position effect was seen. The transgene was expressed specifically in the mammary gland of the transgenic rats. The 210‐kb region is thought to contain all the DNA elements required for proper expression of the human α‐lactalbumin gene. The YAC carrying the human α‐lactalbumin gene is a potential vector for the expression of foreign genes in the mammary gland. Mol. Reprod. Dev. 47:157–163, 1997.

High-level expressing YAC vector for transgenic animal bioreactors.

The position effect is one major problem in the production of transgenic animals as mammary gland bioreactors. In the present study, we introduced the human growth hormone (hGH) gene into 210‐kb human α‐lactalbumin position‐independent YAC vectors using homologous recombination and produced transgenic rats via microinjection of YAC DNA into rat embryos. The efficiency of producing transgenic rats with the YAC vector DNA was the same as that using plasmid constructs. All analyzed transgenic rats had one copy of the transgene and produced milk containing a high level of hGH (0.25–8.9 mg/ml). In transgenic rats with the YAC vector in which the human α‐lactalbumin gene was replaced with the hGH gene, tissue specificity of hGH mRNA was the same as that of the endogenous rat α‐lactalbumin gene. Thus, the 210‐kb human α‐lactalbumin YAC is a useful vector for high‐level expression of foreign genes in the milk of transgenic animals. Mol. Reprod. Dev. 52:414–420, 1999.

Heart rate variability during massive hemorrhage and progressive hemorrhagic shock in dogs

Purpose: To investigate the sequential changes in heart rate (HR), autonomic nervous activity presented by the spectral analysis of heart rate variability (HRV), hemodynamics and metabolism during massive hemorrhage and progressive hemorrhagic shock in dogs.Methods: Twelve dogs were subjected to acute massive hemorrhage until mean arterial pressure (MAP) reached 50 mmHg. Then bleeding was stopped and they were allowed to reach a plateau phase. They were divided, post hoc, into bradycardic or tachycardic groups according to their HR response to the acute massive hemorrhage. After reaching a plateau phase, the dogs were further bled to keep their MAP around 50 mmHg (progressive hemorrhagic shock). Their heart rate power spectra were quantified into low-frequency (LF) (0.04–0.15 Hz) and high-frequency (HF) (0.15–0.4 Hz) components.Results: In the bradycardic group, both LF and HF increased after massive hemorrhage, but during progressive hemorrhagic shock these components decreased while HR increased. In the tachycardic group, LF increased after massive hemorrhage, but during progressive hemorrhagic shock LF decreased with continuous suppression of HF.Conclusion: Massive hemorrhage caused two types of HR response: bradycardia and tachycardia. The HRV profile showed differential autonomic characteristics, and could be a valuable tool in assessing various degrees of hemorrhagic shock.RésuméObjectif: Examiner les changements de fréquence cardiaque (FC), l’activité nerveuse autonome selon l’analyse spectrale de la variabilité de la fréquence cardiaque (VFC), l’hémodynamie et le métabolisme pendant une hémorragie massive et un choc hémorragique progressif, chez des chiens.Méthode: Douze chiens ont été soumis à une hémorragie aiguë massive jusqu’à ce que la tension artérielle moyenne (TAM) atteigne 50 mmHg. Puis, on a arrêté le saignement et laissé la pression parvenir à un plateau. On a, en conséquence, réparti les animaux en groupe bradycardie ou tachycardie selon le compotement de la FC pendant l’hémorragie aiguë massive. Un plateau une fois atteint, les chiens ons subi une autre hémorragie pour amener leur TAM autour de 50 mmHg (choc hémorragique progressif). Le spectre de la puissance de la fréquence cardiaque a été quantifié en composantes de basses fréquences (BF) (0,04–0,15 Hz) et de hautes fréquences (HF) (0,15–0,4 Hz).Résultats: Dans le groupe bradycardie, les BF et HF ont augmenté après l’hémorragie massive, mais lors du choc hémorragique progressif, ces composantes ont diminué pendant que la FC augmentait. Dans le groupe tachycardie, les BF ont augmenté après l’hémorragie massive, mais lors du choc, elles ont baissé en même temps que survenait la suppression continue des HF.Conclusion: L’hémorragie massive a causé deux types de réaction de la FC: la bradycardie et la tachycardie. Le profil de VFC a affiché des caractéristiques autonomes différentielles, ce qui en fait un outil valable pour évaluer différents degrés de choc hémorragique.

Analysis of control elements for position-independent expression of human α-lactalbumin YAC

A major problem in the production of transgenic animal bioreactors using microinjections is the low production rate of high‐expressing transgenic animals due to the position effect. We previously reported that transgenic rats carrying the 210 kb yeast artificial chromosome (YAC) including the human α‐lactalbumin gene express the transgene in a position‐independent manner. The 210 kb YAC was thought to have all the elements necessary for position‐independent expression. In this paper, we constructed fragmented YAC clones and a cosmid clone, and produced transgenic rats to analyze these elements. Transgenic rats with both the 50 kb upstream and downstream regions of the α‐lactalbumin gene had position‐independent expression. Transgenic rats with the 20 kb upstream and downstream regions, however, had position‐dependent expression. Therefore, all the elements necessary for position‐independent expression are thought to be located in the 50 kb upstream to 50 kb downstream region of the α‐lactalbumin gene. Furthermore, we replaced the human α‐lactalbumin promoter with the bovine αS1‐casein promoter in the 210 kb YAC and produced transgenic rats. Position‐dependent expression was observed. The elements required for position‐independent expression of the bovine αS1‐casein gene are different from those required for the human α‐lactalbumin gene, despite the fact that the two genes have the same tissue and developmental specificity. Mol. Reprod. Dev. 54:17–23, 1999.

Gene structure of Bombyx mori larval serum protein (BmLSP)

To understand the molecular mechanisms of the larval‐specific transcription of Bombyx mori larval serum protein (BmLSP), we isolated a clone of the BmLSP gene from a genomic library and sequenced a 3.5‐kb fragment. An intron was found in the 5′ noncoding region of the BmLSP gene. A putative transcription start point was determined by primer extension analysis. Genomic Southern hybridization showed that there is one copy of the BmLSP gene in a haploid genome. A database search revealed that the BmLSP gene has presumptive repetitive sequences found in other B. mori genes, the sequence homologous to ecdysone‐responsive elements and a heptamer sequence found in storage protein genes.

Purification, characterization and developmental changes in the titer of a new larval serum protein of the silkworm, Bombyx mori

Abstract A new protein was found as a major component of hemolymph proteins up to day 1 of the last larval instar of Bombyx mori , and was named Bombyx mori larval serum protein (BmLSP). The BmLSP was purified to homogeneity by ammonium sulfate precipitation, CM-cellulose column chromatography and gel permeation chromatography. The molecular weight of BmLSP was estimated to be 30,000 by SDS-PAGE and 25,000 by gel permeation chromatography. The amino acid composition of BmLSP was similar to that of 30 kDa proteins which are the major serum proteins in the older last (fifth) instar larvae. The 20 NH 2 -terminal amino acids were sequenced and found to be quite different from those of the 30 kDa proteins. Developmental changes in BmLSP titer were followed throughout post-embryonic life by Western blotting using a specific antiserum against BmLSP. Within 1 day after larval hatching, BmLSP appeared in the hemolymph and remained at an almost constant level until day 1 of the last instar. On day 2 of the last instar, the BmLSP level suddenly fell and then gradually decreased toward larval-pupal metamorphosis. Thus, BmLSP is a true larval serum protein and is different from proteins stored for metamorphosis.

A larval serum protein of the silkworm, Bombyx mori : cDNA sequence and developmental specificity of the transcript

The Bombyx mori larval serum protein (BmLSP) is a major component of larval hemolymph proteins until early in the last instar. The cDNA for BmLSP was cloned from a library constructed from fat body RNA of penultimate instar larvae, and the complete nucleotide sequence of the 909 base pair cDNA insert was determined. The deduced 262 amino acid polypeptide included a 16 amino acid residue signal peptide and a 15 amino acid sequence prosegment. A homology search showed that BmLSP has significant similarity with microvitellogenin of Manduca sexta and the 30K proteins of B. mori. Tissue distribution and developmental profile of BmLSP mRNA were analyzed by northern hybridization. BmLSP mRNA was abundant in fat body but not detected in midgut and silk gland. BmLSP mRNA was present during the feeding periods of the fourth and fifth instar larvae, but absent during the larval molt and after the onset of cocoon spinning.

Analysis of the flanking regions of the human α-lactalbumin gene responsible for position-effect independent expression

Transgenic rats with the 130 kb bacterial artificial chromosome construct bLA, including the alpha-lactalbumin gene, had position-independent and copy number-dependent expression, which confirmed previous experiments using the 210 kb yeast artificial construct, yLALBA. To identify elements that confer a position effect, we compared the yLALBA and bLA sequences. yLALBA was chimeric. A common 32 kb region was identified and the total nucleotide sequence was determined. We previously analyzed transgenic rats using polymerase chain reaction to compare the integrity and expression of the transgenes. The -6 to +9 kb region is considered to be necessary for position-independent expression. Transgenic rats lacking the -3.4 to -0.85 kb region had a severe position effect. This 2.5 kb region contains two DNaseI hypersensitive sites at -1.0 and -2.8 kb. The 2.5 kb region is proposed to be a locus control region of the human alpha-lactalbumin gene.

Effect of halothane on intercellular adhesion molecule-1 (ICAM-1) in melanoma cells.

There have only been a few reports relating to the effect of inhalational anesthetics on the tumor cell morphology in cancer patients undergoing surgery. We hypothesized that some anesthetic agents might influence the spread of unresectable cancer cells and might additionally worsen the condition of the patient due to depressed host immune surveillance. We therefore evaluated the influence of halothane on tumor cell adhesion, which is closely linked to tumor cell metastasis. Human melanoma cells from SK-MEL-37 cell-line were exposed to 4% halothane for 3, 6, 12 or 24 hours, respectively. Furthermore, after 24 hours halothane exposure, they were incubated in a 5% CO2 atmosphere for 12 or 24 hours. The cells were then analyzed using a fluorescence flowcytometer and intercellular adhesion molecule-l (ICAM-l) expression in SK-MEL-37 cells was quantified as the intensity of fluorescence of ICAM-l expressed in 10,000 cells. ICAM-l expression in cells exposed to halothane for 3, 6, 12 or 24 hours was lower than that of non-exposed cells and returned to control level after further incubation in 5% CO2 atmosphere for either 12 or 24 hours. We conclude that halothane might affect the progression of tumor cell metastasisin vitro.

Transfer function analysis of the circulation in patients undergoing sevoflurane anesthesia

PurposeThe effects of sevoflurane anesthesia on the interactions between heart rate, blood pressure and respiration were assessed using transfer function analysis.MethodsNine ASA 1 or 2 patients undergoing elective surgery were involved. They were paralysed and their lungs were mechanically ventilated during sevoflurane anesthesia. Instantaneous heart rate (IHR) from electrocardiogram, instantaneous lung volume (ILV) by respiratory inductive plethysmography and mean blood pressure (MBP) by arterial tonometry were obtained during conscious state, and 1 MAC and 2MAC of sevoflurane anesthesia. Transfer function analysis for the relationships between ILV and IHR, ILV and MBP, MBP and IHR were made for five minute periods during which the respiratory rate was varied in a standardized fashion.ResultsIn awake patients transfer magnitudes for the relationships between ILV and IHR and between MBP and IHR in the 0.04–0.5Hz frequency band were 8.9 ± 7.7 bpm·l−1 and 0.95 ± 0.44 bpm·mmHg−1 respectively. Sevoflurane 2MAC decreased these values to 1.2 ± 0.7 (P = 0.014) and 0.26 ± 0.14 (P < 0.01) respectively, but phases were not affected. Neither transfer magnitudes nor phases between ILV and MBP were affected during sevoflurane anesthesia. Coherence for the relationships between ILV and IHR and between MBP and IHR were decreased during 1MAC sevoflurane anesthesia but not affected during 2MAC sevoflurane anesthesia.ConclusionsThe interactions between heart rate, blood pressure and respiration were altered by sevoflurane anesthesia. These findings could be explained by the attenuation of autonomic nervous system activity.RésuméObjectifÉvaluer les effets de l’anesthésie avec du sévoflurane sur les interactions entre la fréquence cardiaque, la tension artérielle et la respiration par l’analyse de la fonction de transfert.MéthodeNeuf patients de classe I ou II ASA, devant subir une intervention élective, ont été recrutés. On les a insensibilisés et placés sous ventilation mécanique pendant l’anesthésie au sévoflurane. On a obtenu la fréquence cardiaque instantanée (FCI), de l’électrocardiogramme; le volume pulmonaire instantané (VPI), de la pléthysmographie respiratoire inductive; la tension artérielle moyenne (TAM), de la tonométrie artérielle, à l’état de conscience, puis à 1CAM et à 2CAM de sévoflurane. L’analyse de la fonction de transfert a été faite pour étudier les relations entre le VPI et la FCI, le VPI et la TAM, la TAM et la FCI pour des périodes de cinq minutes durant lesquelles le rythme respiratoire était modifié d’une façon standard.RésultatsChez les patients éveillés, l’ampleur du transfert pour les relations entre le VPI et la FCI, et entre la TAM et la FCI, selon la bande de fréquences de 0,04–0,5 Hz a été de 8,9 ± 7,7 bpm·l−1 et de 0,95 ± 0,44 bpm·mmHg−1 respectivement. Sous une concentration de 2CAM de sévoflurane, ces valeurs ont baissé à 1,2 ± 0,7 (P = 0,014) et à 0,26 ± 0,14 (P < 0,01) respectivement, mais les phases n’ont pas changé. Ni l’ampleur du transfert, ni les phases entre le VPI et la TAM n’ont été modifiées pendant l’anesthésie au sévoflurane. La cohérence de la relation entre le VPI et la FCI et entre la TAM et la FCI a diminué pendant l’anesthésie avec 1 CAM de sévoflurane, mais n’a pas été modifiée pendant l’utilisation de 2CAM.ConclusionLes interactions entre la fréquence cardiaque, la tension artérielle et la respiration ont été modifiées par l’anesthésie au sévoflurane. Ces résultats peuvent s’expliquer par la réduction de l’activité du système nerveux autonome.