Augmented lipopolysaccharide-induction of the histamine-forming enzyme in streptozotocin-induced diabetic mice

Disorders of the microcirculation and reduced resistance to infection are major complications in diabetes. Histamine enhances capillary permeability, and may also reduce cellular immunity. Here we demonstrate that streptozotocin (STZ)-induced diabetes in mice not only enhances the activity of the histamine-forming enzyme, histidine decarboxylase (HDC), but also augments the lipopolysaccharide (LPS)-induced elevation of HDC activity in various tissues, resulting in a production of histamine. The augmentation of HDC activity occurred as early as 2 days after STZ injection, but was not seen in nondiabetic mice. When given to STZ-treated mice, nicotinamide, an inhibitor of poly(ADP-ribose) synthetase, reduced both the elevation of blood glucose and the elevations of HDC activity and histamine production. These results suggest that hyperglycemia may initiate a sequence of events leading not only to an enhancement of basal HDC activity, but also to a sensitization of mice to the HDC-inducing action of LPS. We hypothesize that bacterial infections and diabetic complications may mutually exacerbate one another because both involved an induction of HDC.     

Critical intracellular Ca2+ concentration for all-or-none Ca2+ spiking in single smooth muscle cells.

Neurotransmitters induce contractions of smooth muscle cells initially by mobilizing Ca2+ from intracellular Ca2+ stores through inositol 1,4,5-trisphosphate (InsP3) receptors. Here we studied roles of the molecules involved in Ca2+ mobilization in single smooth muscle cells. A slow rise in cytoplasmic Ca2+ ([Ca2+]i) in agonist-stimulated smooth muscle cells was followed by a wave of rapid regenerative Ca2+ release as the local [Ca2+]i reached a critical concentration of approximately 160 nM. Neither feedback regulation of phospholipase C nor caffeine-sensitive Ca(2+)-induced Ca2+ release was found to be required in the regenerative Ca2+ release. These results indicate that Ca(2+)-dependent feedback control of InsP3-induced Ca2+ release plays a dominant role in the generation of the regenerative Ca2+ release. The resulting Ca2+ release in a whole cell was an all-or-none event, i.e. constant peak [Ca2+]i was attained with agonist concentrations above the threshold value. This finding suggests a possible digital mode involved in the neural control of smooth muscle contraction.     

A human/mouse chimeric monoclonal antibody against CA125 for radioimmunoimaging of ovarian cancer

Murine monoclonal antibody 196-14 recognizes the ovarian-cancer-associated antigen CA 125, but the epitope it recognizes is different from that of monoclonal antibody OC125. We developed a human/mouse chimeric 196-14 using the variable regions of the murine 196-14 and human heavy-chain (l) and light-chain () constant regions. Cell binding and competitive inhibition assays using chimeric 196-14 labeled with¹²⁵I,¹¹¹In or^99mTc demonstrated that the in vitro immunoreactivity of the chimeric antibody was identical to that of the parental murine monoclonal antibody. However, in mice bearing human ovarian cancer xenografts, the clearance from blood was faster and absolute levels of accumulation in the tumor were lower for the¹²⁵I-labeled or^99mTc-labeled chimeric antibody than for the murine antibody labeled with the corresponding radionuclides. The tumor-to-blood radioactivity ratio was not significantly different between the chimeric antibody and the murine antibody, regardless of the radionuclide used for labeling. Chimeric antibody 196-14 labeled with¹³¹I,¹¹¹In or^99mTc is promising for the radioimmunoimaging of ovarian cancer.     

Activation of phospholipase D by prostaglandin F2α in rat luteal cells and effects of inhibitors of arachidonic acid metabolism

In rat luteal cells labeled with (³H]oleic acid, PGF_2α-stimulated phospholipase D (PLD) activation was investigated. The PLD activity was detected by measuring the accumulation of [³H]phosphatidylethanol (PtdEt) in the presence of ethanol. PGF_2α stimulated PtdEt accumulation at concentrations of more than 100 nM in the presence of ethanol. However, PtdEt accumulation did not change in the absence of ethanol. PGF_2α (1 μM) increased PtdEt accumulation after 1 min, and the accumulation reached a plateau by 2–3 min. These results indicate that PGF_2α activates PLD in rat luteal cells. U-73122, a phospholipase C (PLC) inhibitor, and staurosporine, a protein kinase C (PKC) inhibitor, did not inhibit PGF_2α-stimulated [³H]PtdEt accumulation. These results suggest that PGF_2α-induced PLD activation is different from PLC-PKC systems. We reported previously that PGF_2α stimulated the release of arachidonic acid. The effects of indomethacin, nordihydroguaiaretic acid (NDGA), and 5,8,11,14-eicosatetraynoic acid (ETYA), inhibitors of arachidonic acid metabolism, on PGF_2α-stimulated PtdEt accumulation were examined. Pretreatment with indomethacin enhanced PGF_2α-induced PtdEt accumulation. In contrast, pretreatment with NDGA and ETYA inhibited PGF_2α-induced PtdEt accumulation. It is suggested that PGF_2α-stimulated PLD activation is mediated via lipoxygenase products.     

Mutation and polymorphism analysis of the TRKA (NTRK1) gene encoding a high-affinity receptor for nerve growth factor in congenital insensitivity to pain with anhidrosis (CIPA) families

The human TRKA gene encodes a high-affinity tyrosine kinase receptor for nerve growth factor. Congenital insensitivity to pain with anhidrosis (CIPA) is an autosomal recessive genetic disorder reported from various countries and characterized by anhidrosis (inability to sweat), the absence of reaction to noxious stimuli, and mental retardation. We have found that TRKA is the gene responsible for CIPA. We have studied TRKA in 46 CIPA chromosomes derived from 23 unrelated Japanese CIPA families. including three that have been previously reported, and identified 11 novel mutations. Four (L93P, G516R, R648 C, and D668Y) are missense mutations that result in amino acid substitutions at positions conserved in the TRK family, including TRKA, TRKB, and TRKC. Three (S131 fs, L579 fs, and D770 fs) are frameshift mutations. Three (E164X, Y359X, and R596X) are nonsense mutations. The other is an intronic branch-site (IVS7-33T-->A) mutation, causing aberrant splicing in vitro. We also report the characterization of eight intragenic polymorphic sites, including a variable dinucleotide repeat and seven single nucleotide polymorphisms, and describe the haplotypic associations of alleles at these sites in 106 normal chromosomes and 46 CIPA chromosomes. More than 50% of CIPA chromosomes share the frameshift mutation (R548 fs) that we described earlier. This mutation apparently shows linkage disequilibrium with a rare haplotype in normal chromosomes, strongly suggesting that it is a common founder mutation. These findings represent the first extensive analysis of CIPA mutations and associated intragenic polymorphisms; they should facilitate the detection of CIPA mutations and aid in the diagnosis and genetic counseling of this painless but severe genetic disorder with devastating complications.     

Mouse Shh is required for prechordal plate maintenance during brain and craniofacial morphogenesis

In humans, holoprosencephaly (HPE) is a common birth defect characterized by the absence of midline cells from brain, facial, and oral structures. To understand the pathoetiology of HPE, we investigated the involvement of mammalian prechordal plate (PrCP) cells in HPE pathogenesis and the requirement of the secreted protein sonic hedgehog (Shh) in PrCP development. We show using rat PrCP lesion experiments and DiI labeling that PrCP cells are essential for midline development of the forebrain, foregut endoderm, and ventral cranial mesoderm in mammals. We demonstrate that PrCP cells do not develop into ventral cranial mesoderm in Shh^−/− embryos. Using Shh^−/− and chimeric embryos we show that Shh signal is required for the maintenance of PrCP cells in a non-cell autonomous manner. In addition, the hedgehog (HH)-responding cells that normally appear during PrCP development to contribute to midline tissues, do not develop in the absence of Shh signaling. This suggests that Shh protein secreted from PrCP cells induces the differentiation of HH-responding cells into midline cells. In the present study, we show that the maintenance of a viable population of PrCP cells by Shh signal is an essential process in development of the midline of the brain and craniofacial structures. These findings provide new insight into the mechanism underlying HPE pathoetiology during dynamic brain and craniofacial morphogenesis.     

Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4T cells

CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.     

Asymmetric Wavefront Aberrations and Pupillary Shapes Induced by Electrical Stimulation of Ciliary Nerve in Cats Measured with Compact Wavefront Aberrometer

To investigate the changes in the wavefront aberrations and pupillary shape in response to electrical stimulation of the branches of the ciliary nerves in cats. Seven eyes of seven cats were studied under general anesthesia. Trains of monophasic pulses (current, 0.1 to 1.0 mA; duration, 0.5 ms/phase; frequency, 5 to 40 Hz) were applied to the lateral or medial branch of the short ciliary nerve near the posterior pole of the eye. A pair of electrodes was hooked onto one or both branch of the short ciliary nerve. The electrodes were placed about 5 mm from the scleral surface. The wavefront aberrations were recorded continuously for 2 seconds before, 8 seconds during, and for 20 seconds after the electrical stimulation. The pupillary images were simultaneously recorded during the stimulation period. Both the wavefront aberrations and the pupillary images were obtained 10 times/sec with a custom-built wavefront aberrometer. The maximum accommodative amplitude was 1.19 diopters (D) produced by electrical stimulation of the short ciliary nerves. The latency of the accommodative changes was very short, and the accommodative level gradually increased up to 4 seconds and reached a plateau. When only one branch of the ciliary nerve was stimulated, the pupil dilated asymmetrically, and the oblique astigmatism and one of the asymmetrical wavefront terms was also altered. Our results showed that the wavefront aberrations and pupillary dilations can be measured simultaneously and serially with a compact wavefront aberrometer. The asymmetric pupil dilation and asymmetric changes of the wavefront aberrations suggest that each branch of the ciliary nerve innervates specific segments of the ciliary muscle and dilator muscle of the pupil.     

Generation of Rodent Malaria Parasites with a High Mutation Rate by Destructing Proofreading Activity of DNA Polymerase δ

Plasmodium falciparum malaria imposes a serious public health concern throughout the tropics. Although genetic tools are principally important to fully investigate malaria parasites, currently available forward and reverse tools are fairly limited. It is expected that parasites with a high mutation rate can readily acquire novel phenotypes/traits; however, they remain an untapped tool for malaria biology. Here, we generated a mutator malaria parasite (hereinafter called a ‘malaria mutator’), using site-directed mutagenesis and gene transfection techniques. A mutator Plasmodium berghei line with a defective proofreading 3′ → 5′ exonuclease activity in DNA polymerase δ (referred to as PbMut) and a control P. berghei line with wild-type DNA polymerase δ (referred to as PbCtl) were maintained by weekly passage in ddY mice for 122 weeks. High-throughput genome sequencing analysis revealed that two PbMut lines had 175–178 mutations and a 86- to 90-fold higher mutation rate than that of a PbCtl line. PbMut, PbCtl, and their parent strain, PbWT, showed similar course of infection. Interestingly, PbMut lost the ability to form gametocytes during serial passages. We believe that the malaria mutator system could provide a novel and useful tool to investigate malaria biology.