The water channel gene in human uterus

The cDNA coding the water channel was isolated from a human uterus cDNA library template by a one-step polymerase chain reaction (PCR). The oligonucleotide primers corresponding to the 5' untranslated nucleotide sequence and complementary to the 3' untranslated nucleotide sequence of the cDNA coding the 28 kDa erythrocyte integral membrane protein (CHIP28) were synthesized and used to initiate the reaction. A 1340 bp cDNA coding the human uterine water channel (hUWC) was cloned and sequenced. The hUWC showed 99.8% and 99% identity with the nucleotide and amino-acid sequences of CHIP28, respectively. The deduced hUWC polypeptide is composed of 269 amino acid residues with a single amino acid variant from CHIP28 protein at position 45, where valine replaces alanine. The hUWC cDNA translated in a prokaryotic protein expression system produced a protein with an estimated Mr of 28 kDa, equivalent to the size of the human red cell CHIP28 protein. The present results suggest that the human uterus contains water channels that may play an important role in regulating water transport and imbibition in the uterus.     

Identification of protein A-binding components in Spisula oocytes

Components involved in sustaining meiosis arrest of oocytes were determined. Proteins that bind to protein A from meiosis-arrested and 5-HT-matured Spisula oocytes were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Meiosis-arrested oocytes contained three doublets of proteins with estimated Mrs of 43 and 45, 38 and 40, and 21 and 23 kDa. In 5 HT-matured oocytes the 21 and 23 and 38 and 40 kDa proteins were retained; whereas the 43 and 45 kDa proteins were absent. The protein A-bound proteins did not interact with antibodies against the various subclasses of human, mouse, rat and rabbit IgG or human Fc fragment. The amino acid sequence of the N-terminus of the 43 kDa protein was determined to be NH2-VLRIGSGMXDT. Comparison of this sequence with existing database at Protein Identification Resource (R 32.0), GenBank (R 72.0), SWISS-PROT (R 22.0), and EMBL (R 31.0) showed no homology with any reported protein. The protein A-bound components from meiosis-arrested oocytes were incubated in vitro with [gamma-32P]ATP. Only the 68 kDa protein was radiophosphorylated. This protein was not detected in 5-HT-matured oocytes. The disappearance of the 43, 45, and 68 kDa proteins in 5-HT-matured oocytes suggests that these components may be involved in sustaining meiosis meiosis. A unique property of these proteins is that they interact with protein A and are distinctly different from immunoglobulin.     

Design of Synthetic Polymer Nanoparticles that Capture and Neutralize a Toxic Peptide

Designed polymer nanoparticles (NPs) capable of binding and neutralizing a biomacromolecular toxin are prepared. A library of copolymer NPs is synthesized from combinations of functional monomers. The binding capacity and affinity of the NPs are individually analyzed. NPs with optimized composition are capable of neutralizing the toxin even in a complex biological milieu. It is anticipated that this strategy will be a starting point for the design of synthetic alternatives to antibodies.     

In vitro assessment of the cytotoxicity of anti-allergic eye drops using 5 cultured corneal and conjunctival cell lines

The aim of this study was to evaluate the cytotoxicity of anti-allergic eye drops for human corneal endothelial cells (HCEC) and commercially available ocular surface cells. A primary HCEC culture was derived from human eye bank specimens. SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells) were obtained commercially. The WST-1 assay was used to measure HCEC viability, and the viability of other cells was measured using the MTT assay. Cells were treated with 7 commercially available anti-allergic eye drops for 48 h and cell viability was measured and calculated as a percentage of control. The degree of toxicity for each eye-drop solution was based on the cell viability score (CVS). HCECs treated with a 1000-fold dilution of the eye-drop solution had a viability score of 67% for Rizaben and ≥80% for the other solutions with Zepelin being the least toxic. Cell viability in response to eye-drop solutions preserved with benzalkonium chloride (BAK) was dependent on the concentration of the drug solution and exposure time. Treatment of ocular surface cells with a 20-fold dilution of the eye-drop solution resulted in the following order of cell viability as determined by their CVS: Zepelin > Ketas = Zaditen ≥ Tramelas PF = Patanol ≥ Rizaben ≥ Livostin. This order was similar to that observed for HCECs, and cell viability was found to be concentration-dependent. Based on the penetration of the drug into eye tissues, HCECs are only likely to be pharmaceutically damaging in rare cases. Epithelial cell viability depends primarily on the concentration of BAK rather than on the action of the active component in the eye-drop solution. CVS values were useful for comparison of toxicity.     

Metal salts requisite for the production of eicosapentaenoic acid by a marine bacterium isolated from mackerel intestines

Metal salts important for the growth and 5,8,11,14,17- ciseicosapentaenoic acid EPA) production of a bacterium isolated from Pacific mackerel intestines were investigated at 25°C in culture media containing 1.0% peptone and 0.50% yeast extract, and the composition of an optimum culture medium was determined. This bacterium could grow in the media in which sodium chloride was the sole added inorganic component. By raising the concentration of sodium chloride from 1.2 to 2.4%, the yield of bacterial cells increased and the yield of EPA reached a maximum at 2.0% NaCl concentration. In contrast to calcium chloride, potassium chloride and magnesium chloride as second metal salts promoted the growth of this bacterium at relatively low concentrations without inhibiting the accumulation of EPA. The yield of EPA reached its maximum value of 51.9 mg/L of culture broth at 8 hr at 2.0% NaCl, 0.15% KCl and 0.16% MgCl₂ concentrations. This yield of EPA was 20% greater than that obtained with Jamarin S artificial sea water. *To whom correspondence should be addressed at Department of Chemical Engineering, Tokyo Institute of Technology, 2-12-1 Ookayama, Meguro-ku, Tokyo 152, Japan. ¹Sagami Chemical Research Center, 4-4-1 Nishi-ohnuma, Sagamihara, Kanagawa 229, Japan.     

NMR studies of Borrelia burgdorferi OspA, a 28 kDa protein containing a single-layer beta-sheet

Viruses such as HIV, influenza, picornavirus and others are known stimulators of apoptosis. This individual cellular elimination is a preferential host defense in regenerative tissues. In contrast, if this death occurred in nonregenerating cells, such as neurons of the central nervous system, may result in disease. The target cell for rabies virus is the neuron. Here we studied the outcome of the interaction between rabies virus (CVS-11) and mouse brain cells. Replication of rabies virus in suckling mouse brain cells resulted in brain cell apoptosis, detected by DNA fragmentation and in situ apoptosis within 25 h after infection and before evidence of intracerebral immune activation. Cell death occurred simultaneously with rabies virus replication. There were clinical signs of illness in infected newborn mice within 24 h after the appearance of DNA fragmentation and before infiltration by lymphocytes. This suggested that onset of illness started independently of the immune function. This conclusion was supported by the occurrence of massive apoptosis followed by paralysis in rabies virus-infected immunosuppressed mice. Direct, viral-induced, neuronal apoptosis was the earliest death mechanism detected in these mice. We propose that pathogenesis of this fixed strain of rabies virus in mice begins with the induction of apoptosis by rabies virus replication. Cerebral damage may then be amplified by immunological mechanisms plus an additional unidentified factor. This is followed by increased permeability of the blood brain barrier.