Human testicular protein TPX1/CRISP-2: localization in spermatozoa, fate after capacitation and relevance for gamete interaction

Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.     

The color reaction between partially saponified poly(vinyl acetate) and iodine-iodide in the presence of amylose

The color reaction between partially saponified poly(vinyl acetate), (P-PVAc), which is dissolvable in water and iodine-iodide was investigated in the presence of amylose. As the concentration of amylose increases, the absorbance at λ_max = 488 nm of the red-violet complex between P-PVAc and iodine-iodide decreases and, on the other hand, the absorbance at λ_max = 625 nm of the blue complex between amylose and iodine-iodide increases. The equation for the relationship between the decrease in the red-violet complex and the increase in the blue complex was derived. On the basis of this equation, the molar absorption coefficient per iodine molecule at λ_max = 625 nm for the blue complex of amylose with iodine-iodide was found to be 3,68·10⁴ l mol^?1 cm^?1 at 15°C.     

Rhythmical contractions in pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats

Rhythmical contractions accompanied by an increase in cytosolic Ca2+ concentrations were produced in ring preparations of endothelium-denuded pulmonary arteries from monocrotaline-treated rats, but not in those from vehicle-treated rats, 2-3 h after a resting tension of 15 mN (150-180% of the initial wall length of the artery) was applied. The rhythmical contractions were abolished by nicardipine and ryanodine. Cyclopiazonic acid reduced the relaxation phase of the rhythmical contractions, finally leading to a sustained contraction. Similarly, apamin caused a sustained contraction, whereas charybdotoxin increased the amplitude of the rhythmical contractions. Glibenclamide had no apparent effects on them. Indomethacin and the prostaglandin H2/thromboxane A2 receptor antagonist SQ29548 abolished the rhythmical contractions and reduced the tension, but the thromboxane synthase inhibitor ozagrel had no effect. These results suggest that optimal stretch induces rhythmical contractions in the pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats, to which both Ca2+ influx through voltage-operated Ca2+ channels and Ca2+ release from the endoplasmic reticulum seem to contribute. It is also suggested that small-conductance Ca(2+)-activated K+ channels participate in the relaxation phase of rhythmical contractions. Furthermore, prostaglandin H2 released from nonendothelial cells is likely to play a pivotal role in the induction of rhythmical contractions.     

Development of a high-level expression system for deuterium-labeled human serum albumin

We have developed an expression system for recombinant human serum albumin (rHSA) using methylotrophic yeast Pichia pastoris Mut(+) transformants together with the multiple cross-over integration of the vector containing human serum albumin (HSA). After 86 h of methanol induction, the secreted rHSA reached levels of approximately 320 mg/l in 100% H(2)O medium and approximately 180 mg/l in 70% D(2)O/30% H(2)O (v/v) medium in a fed-batch fermenter. The structures of the obtained rHSA and plasma-derived HSA (pHSA) were virtually identical as viewed from various physicochemical techniques such as HPLC, SDS gel electrophoresis, and CD. NMR peaks of the partially deuterium (D)-labeled rHSA (DrHSA) were quite sharp compared to those of pHSA due to suppression of the intramolecular nuclear Overhauser effect, promising further structural studies of the whole HSA molecule in the solution state using the recent NMR techniques.     

Development of sutureless vascular connecting system for easy implantation of small-caliber artificial grafts

A novel sutureless vascular connecting system, an assembly with a delivery rod, an introducing sheath, and a connecting device, was developed for easy implantation of small-caliber vascular grafts less than 2 mm in internal diameter. A microporous stainless tube (length 2 mm, external diameter 1.6 mm, wall thickness 65 µm, pore diameter 400 µm, pore-to-pore distance 500 µm) was designed to serve as a connecting device. The feasibility of the system was tested using two types of preliminary animal experiments. One animal model consisted of graft implantation into the rat abdominal aorta (1.5 mm in diameter). The connecting device was inserted into the proximal and distal ends of the aorta through the introducing sheath by pushing the delivery rod with the connecting device placed over it. Subsequently, the aortic segments were inserted into both ends of model grafts made of segmented polyurethane (1.8 mm in internal diameter) and were fixed with banding silk threads from the exterior. The procedure was completed within 20 min without requiring specialized microsurgery techniques. Blood leakage and obstruction did not occur. The second model consisted of an end-to-end anastomosis between rabbit common carotid arteries (2 mm in diameter), which was performed within several minutes of blood flow interruption. Scanning electron microscopy demonstrated that the luminal surface of the device was fully covered with endothelial cells (ECs) after 1 week as a result of transluminal ingrowth of native ECs through the micropores in the device. This endothelialization may prevent early thrombus-induced occlusion. This simple and “easy-to-learn” technique will promote the development of small-caliber arterial grafts, and furthermore, it may have potential for clinical application.     

Immunoelectron microscopic study of HTLV-II-producing cells with an anti-envelope gp46 monoclonal antibody

Summary We observed expression of envelope gp46 protein on an HTLV-II-producing T-cell line (Mo) cells by an immunoelectron microscopical method using a monoclonal antibody against HTLV-II gp46. gp46 reactivity was observed on virus-like particles, extracellular vesicles, cell membrane, and partially in nuclear membrane and endoplasmic reticulum.     

Activity of anthocyanins from fruit extract of Ribes nigrum L. against influenza A and B viruses

Earlier, we have detected antiviral activity in an extract from Ribes nigrum L. fruits ("Kurokarin", name of the one species of black currant in Japanese) against influenza A and B viruses, and herpes simplex virus 1 (Knox et al., Food Processing 33, 21-23, 1998). In the present study, the antiviral activity of constituents of a Kurokarin extract and the mechanism of its antiviral action were examined. Kurokarin extracts were separated to fractions A to D by column chromatography. The major constituents of the fraction D were estimated as anthocyanins. The fraction D was further fractionated by thin-layer chromatography (TLC) to fractions A' to G'. The fraction E' consisted of 3-O-alpha-L-rhamnopyranosyl-beta-D-glucopyranosyl-cyanidin and 3-O-beta-D-glucopyranosyl-cyanidin, and the fraction F' consisted of 3-O-alpha-L-rhamnopyranosyl-beta-D-glucopyranosyl-delphinidin and 3-O-beta-D-glucopyranosyl-delphinidin, identified by high performance liquid chromatography (HPLC) with standards and by high resolution mass spectrometry. The fractions D' to G' showed potent antiviral activity against influenza viruses A and B. The additive antiviral effect of a combination of the fractions E' and F' was assessed. Anthocyanins in the fraction F' did not directly inactivate influenza viruses A and B, but they inhibited virus adsorption to cells and also virus release from infected cells.     

Legionellosis

Although increasing attention is being given to Legionella pneumonia in Japan, reports of solitary onset of this disease are scant in Japan. The patient, from whom L. dumoffii was isolated, was a 59-year-old male with no underlying disease. He visited our hospital because of fever and cough, and was admitted to our department for X-ray findings consistent with pneumonia. After admission, pulmonary lesions spread rapidly, and based on the suspicion of Legionella pneumonia, drugs such as EM, RFP and MINO were used. However, the patient died on the 26th hospital day. L. dumoffii was isolated from specimens obtained by airway aspiration before death and specimens of lung abscess and airway discharge obtained during autopsy (7 specimens in total). In addition, the L. dumoffii antibody titer in the serum became elevated. This is the first case of L. dumoffii pneumonia reported in Japan. The other case was in an 81-year-old male with underlying disease. He was admitted urgently with suspected pneumonia but died on the following day. L. pneumophila serogroup 5 was isolated from autopsied lung tissue. Fatality is high for this disease, making early diagnosis and treatment with appropriate antibiotics essential. Physicians should bear in mind the possibility of this disease and request the necessary laboratory tests in suspected cases without delay.