Tissue-nonspecific alkaline phosphatase with an Asp(289)-->Val mutation fails to reach the cell surface and undergoes proteasome-mediated degradation

A missense mutation in the gene of tissue-nonspecific alkaline phosphatase, which replaces aspartic acid at position 289 with valine [TNSALP (D289V)], was reported in a lethal hypophosphatasia patient [Taillandier, A. et al. (1999) Hum. Mut. 13, 171-172]. To define the molecular defects of TNSALP (D289V), this mutant protein in transiently transfected COS-1 cells was analyzed biochemically and morphologically. TNSALP (D289V) exhibited no alkaline phosphatase activity and mainly formed a disulfide-linked high molecular mass aggregate. Cell-surface biotinylation, digestion with phosphatidylinositol-specific phospholipase C and an immunofluorescence study showed that the mutant protein failed to appear on the cell surface and was accumulated intracellularly. In agreement with this, pulse/chase experiments demonstrated that TNSALP (D289V) remained endo-beta-N-acetyl- glucosaminidase H-sensitive throughout the chase and was eventually degraded, indicating that the mutant protein is unable to reach the medial-Golgi. Proteasome inhibitors strongly blocked the degradation of TNSALP (D289V), and furthermore the mutant protein was found to be ubiquitinated. Besides, another naturally occurring TNSALP with a Glu(218)-->Gly mutation was also found to be polyubiquitinated and degraded in the proteasome. Since the acidic amino acids at positions 218 and 289 of TNSALP are thought to be directly involved in the Ca(2+) coordination, these results suggest the critical importance of calcium binding in post-translational folding and assembly of the TNSALP molecule.     

Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation in the cytokinetic process

Aurora-B is an evolutionally conserved protein kinase that regulates several mitotic events including cytokinesis. We previously demonstrated the possible existence of a protein kinase that phosphorylates at least Ser-72 on vimentin, the most widely expressed intermediate filament protein, in the cleavage furrow-specific manner. Here we showed that vimentin-Ser-72 phosphorylation occurred specifically at the border of the Aurora-B-localized area from anaphase to telophase. Expression of a dominant-negative mutant of Aurora-B led to a reduction of this vimentin-Ser-72 phosphorylation. In vitro analyses revealed that Aurora-B phosphorylates vimentin at approximately 2 mol phosphate/mol of substrate for 30 min and that this phosphorylation dramatically inhibits vimentin filament formation. We further identified eight Aurora-B phosphorylation sites, including Ser-72 on vimentin, and then constructed the mutant vimentin in which these identified sites are changed into Ala. Cells expressing this mutant formed an unusually long bridge-like intermediate filament structure between unseparated daughter cells. We then identified important phosphorylation sites for the bridge phenotype. Our findings indicate that Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation and controls vimentin filament segregation in cytokinetic process.     

Role of microtubules versus myosin heavy chain isoforms in contractile dysfunction of hypertrophied murine cardiocytes

In large mammals there is a correlation between microtubule network densification and contractile dysfunction in severe pressure-overload hypertrophy. In small mammals there is a similar correlation for the shift to beta-myosin heavy chain (MHC), a MHC isoform having a slower ATPase Vmax. In this study, murine left ventricular (LV) pressure overload invoked both mechanisms: microtubule network densification and beta-MHC expression. Cardiac beta-MHC was also augmented without altering tubulin levels by two load-independent means, chemical thyroidectomy and transgenesis. In hypertrophy, contractile function of the LV and its cardiocytes decreased proportionally; microtubule depolymerization restored normal cellular contraction. In hypothyroid mice having a complete shift from alpha-MHC to beta-MHC, contractile function of the LV and its cardiocytes also decreased, but microtubule depolymerization had no effect on cellular contraction. In transgenic mice having a cardiac beta-MHC increase similar to that in hypertrophy, contractile function of the LV and its cardiocytes was normal, and microtubule depolymerization had no effect. Thus, although both mechanisms may cause contractile dysfunction, for the extent of MHC isoform switching seen even in severe murine LV pressure-overload hypertrophy, microtubule network densification appears to have the more important role.     

Desipramine attenuates loss of cardiac sympathetic neurotransmitters produced by congestive heart failure and NE infusion

We reported recently that inhibition of neuronal reuptake of norepinephrine (NE) by desipramine prevented the reduction of sympathetic neurotransmitters in the failing right ventricle of right heart failure animals. In this study, we studied whether desipramine also reduced the sympathetic neurotransmitter loss in animals with left heart failure induced by rapid ventricular pacing (225 beats/min) or after chronic NE infusion (0.5 microg. kg(-1). min(-1)). Desipramine was given to the animals for 8 wk beginning with rapid ventricular pacing or NE infusion. Animals receiving no desipramine were studied as controls. We measured myocardial NE content, NE uptake activity, and sympathetic NE, tyrosine hydroxylase, and neuropeptide Y profiles by histofluorescence and immunocytochemical techniques. Effects of desipramine on NE uptake inhibition were evidenced by potentiation of the pressor response to exogenous NE and reduction of myocardial NE uptake activity. Desipramine treatment had no effect in sham or saline control animals but attenuated the reduction of sympathetic neurotransmitter profiles in the left ventricles of animals with rapid cardiac pacing and NE infusion. In contrast, the panneuronal marker protein gene product 9.5 profile was not affected by either rapid pacing or NE infusion, nor was it changed by desipramine treatment in the heart failure animals. The study confirms that excess NE contributes to the reduction of cardiac sympathetic neurotransmitters in heart failure. In addition, it shows that the anatomic integrity of the sympathetic nerves is relatively intact and that the neuronal damaging effect of NE involves the uptake of NE or its metabolites into the sympathetic nerves.     

Upregulation of mRNA Encoding the M5 Muscarinic Acetylcholine Receptor in Human T- and B-Lymphocytes During Immunological Responses

Lymphocytes possess an independent, non-neuronal cholinergic system. Moreover, both T- and B-lymphocytes express multiple muscarinic acetylcholine receptors (mAChR). To obtain a better understanding of the regulatory mechanisms governing mAChR gene expression in the lymphocytic cholinergic system, we examined the effects of lymphocyte activation on expression of mAChR mRNA. Stimulation of T- and B-lymphocytes, respectively, with T-cell activator phytohemagglutinin and B-cell activator Staphylococcus aureus Cowan I upregulated M₅ mAChR mRNA expression in the CEM human leukemic T-cell line and in the Daudi B-cell line, which served as models of lymphocytes. In striking contrast, M₃ and M₄ mAChR mRNA expression was not affected in either cell line. Nonetheless, stimulating lymphocytes with phorbol 12-myristate 13-acetate, a protein kinase C activator, plus ionomycin, a calcium ionophore, upregulated expression of both M₃ and M₅ mAChR mRNA. This represents the first demonstration that immunological stimulation leads to M₅ mAChR gene expression in lymphocytes.     

Analysis of the flanking regions of the human alpha-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.     

The NB protein of influenza B virus is not necessary for virus replication in vitro

The NB protein of influenza B virus is thought to function as an ion channel and therefore would be expected to have an essential function in viral replication. Because direct evidence for its absolute requirement in the viral life cycle is lacking, we generated NB knockout viruses by reverse genetics and tested their growth properties both in vitro and in vivo. Mutants not expressing NB replicated as efficiently as the wild-type virus in cell culture, whereas in mice they showed restricted growth compared with findings for the wild-type virus. Thus, the NB protein is not essential for influenza B virus replication in cell culture but promotes efficient growth in mice.     

Production of recombinant human relaxin 3 in AtT20 cells

Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 microM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.     

Simple maxillary reconstruction using free tissue transfer and prostheses

Prosthetic rehabilitation is essential for maintaining postoperative oral function after maxillary reconstruction. However, the maxillary prosthesis becomes unstable in some patients because of extensive palatomaxillary resection and drooping of the transferred flap. In such patients, maintaining sufficient oral function is difficult, especially if the patient is edentulous. To achieve prosthetic retention, the authors performed microvascular maxillary reconstruction with a slit-shaped fenestration in the midline of the hard palate. Maxillary defects after subtotal or total maxillectomy were reconstructed with rectus abdominis musculocutaneous flaps in five patients. Defects of the nasal lining and palate were reconstructed with the single cutaneous portion of the flap, and a slit-shaped fenestration was left between the cutaneous portion of the flap and the edge of the remaining hard palate. Postoperatively, patients were fitted with maxillary prostheses that had a flat projection for the palatal fenestration. In all patients, the prosthesis was stable enough for mastication and prevented nasal regurgitation. Speech function was rated as excellent on Hirose's scoring system for Japanese speech ability. The authors believe that their method of palatomaxillary reconstruction is both simple and reliable.