The effect of hydrophilic adhesive monomers on the stability of type I collagen

The adsorption effects of adhesive monomers on the structural stability of type I collagen were studied at an acid pH condition for two monomers: 2-hydroxyethyl methacrylate (HEMA), a neutral monomer and N-methacryloyl glycine (NMGly), an acidic monomer. Differential scanning calorimetry (DSC) measurements were done to assess the denaturation temperature (Td), which is a measure of the structural stability of the proteins, including the bovine tendon collagen (BTC). While HEMA lowered the Td of the BTC linearly with HEMA concentrations, NMGly exhibited a two-step decrease of the Td. The rate of decrease in the Td by the NMGly was by far greater than the rate of decrease with the HEMA. The first step had a larger slope than the second step in the Td vs. CNMGly plot. The degree of adsorption of these two monomers to the BTC was estimated from infrared absorption measurements on the monomer solutions of various concentrations, before and after the immersion of the BTC. Both the adsorption of HEMA to the BTC and the Td of the BTC were linearly dependent on HEMA concentrations. Conversely, NMGly was adsorbed to the BTC, again, in a two-step decrease similar to the Td vs. CNMGLy profile. An enhanced adsorption of NMGly, which might be attributed to a strong electrostatic interaction, was observed below 0.013 mol%. Circular dichroism measurements of the collagen of the same type as the BTC, in the absence and in the presence of the monomers, revealed that the native collagen helix structure was scarcely affected by the monomers. From these observations, it was concluded that (1) both of the monomers were adsorbed onto the BTC, which thus destabilized the triple helical collagen structure, and that (2) the effect was higher for NMGly in which the electrostatic attraction with the oppositely charged collagen might be effective at a pH of 3. If compared to HEMA, an acidic NMGly is a potential monomer that binds strongly to collagen and one that is hardly hydrolyzed.     

A trial to restore defective human sperm centrosomal function

BACKGROUND: In human fertilization, sperm centrosome function is essential for male and female pronuclear movement and fusion. In this study, we investigated the possibility of restoring human sperm centrosomal function in sperm exhibiting abnormalities in microtubule organization. METHODS: Semen was obtained from both a fertile donor and a patient with dysplasia of the fibrous sheath (DFS). Following heterologous ICSI using human sperm, we examined microtubules and chromatin configuration in bovine oocytes. Sperm were treated with dithiothreitol (DTT) prior to ICSI, while the oocytes were treated with the cytoskeletal stabilizer paclitaxel after ICSI. RESULTS: The combination of DTT and paclitaxel treatment induced microtubule organization in dead sperm from the fertile donor following heterologous ICSI. This treatment, however, was not effective for DFS sperm. In addition, expression of centrin, a protein functioning within the sperm centrosome, was reduced in DFS sperm from that of the normal levels observed in fertile donor sperm. CONCLUSION: These results indicate that sperm centrosomal function could be induced by the treatment of sperm with DTT before ICSI and of oocytes with paclitaxel after ICSI. DFS sperm are likely to exhibit such severe dysfunction of sperm centrosome that cannot be compensated for by this treatment; therefore, this method may be a practical way to discern the degree of sperm centrosomal dysfunction.     

Expression profiles of various transporters for oligopeptides, amino acids and organic ions along the human digestive tract

Various transporters such as H⁺/peptide cotransporter PEPT1 are expressed in the intestine, and play important physiological and pharmacological roles in the body. Present study was performed to examine the expression profile of 20 kinds of transporters (PEPT1 and 2, P-glycoprotein, amino acid transporters and organic ion transporters) along the human digestive tract, especially focusing on PEPT1. Using normal mucosal specimens, real-time polymerase chain reactions were carried out. Immunoblot analyses were also performed for PEPT1 expression. PEPT1 mRNA was highly expressed in the small intestine (duodenum > jejunum > ileum) compared to other tissues, and some patients showed a significant level of expression in the stomach. The expressional pattern of PEPT1 in the stomach and histological diagnosis indicated that gastric PEPT1 originated from the intestinal metaplasia. The amino acid transporters showed unique mRNA expression levels and distributions in the digestive tract. For example, the expression levels of B⁰AT1, a Na⁺-dependent and chloride-independent neutral amino acid transporter, were increased from the duodenum to ileum, which pattern is completely inverted to that for PEPT1. There is little expression of organic ion transporters except for organic cation/carnitine transporter OCTN2. In conclusion, PEPT1 was abundantly expressed in the small intestine, and the reciprocal expression of PEPT1 and B⁰AT1 may serve for the efficient absorption of protein digestive products.     

Immunoelectron microscopic localization of protein 4.1B in proximal S1 and S2 tubules of rodent kidneys

Protein 4.1 family proteins are supposed to interact with intramembranous proteins and membrane skeletons. Protein 4.1B, one of the family proteins, was recently reported to be localized in basolateral regions of mouse renal proximal tubules. In this study, we extended the idea that protein 4.1B may be related to ion balance in the region by immunohistochemical studies by light and electron microscopy with our antibody against protein 4.1B. Protein 4.1B distribution in rodent kidneys was determined by comparing with lectin Lotus tetragonobulus agglutinin (LTA), a proximal tubule cell marker, and also with Na⁺/HCO₃^–-cotransporter-1 (NBC-1), being expressed in basolateral domains of segment S1 to S2 proximal tubule epithelial cells. Specific protein 4.1B immunolabeling was observed in cuboidal epithelial cells basolaterally, starting their localization from a urinary pole at the glomerulus, whereas the squamous epithelial cells of Bowmans capsules were immunonegative. Rat Bowmans capsules had no simple cuboidal cells, where no protein 4.1B immunostaining appeared. All the protein 4.1B-positive epithelial cells were LTA positive. By immunoelectron microscopy, protein 4.1B immunolabeling of the proximal epithelial cells was restricted to the basolateral membranes, including basal infoldings, whereas tight junctions were not immunolabeled. It is concluded that protein 4.1B might play a role related to membrane skeletal proteins in the basolateral membranes of S1 and S2 proximal tubule cells. Moreover, the immunolocalization of protein 4.1B was almost the same as that of NBC-1, indicating a possible function as a regulator of ion balance, such as Na⁺ and HCO₃^– reabsorption.     

Observation of tongue papillae by video microscopy and contact endoscopy to investigate their correlation with taste function

OBJECTIVES: Contact endoscopy is a technique used to obtain detailed images of living epithelium in the fields of gynecology, rhinology and laryngology. Video microscopy is useful for observation of the surface of tongue papillae. In the present study, we attempted to apply the contact endoscopy technique together with video microscopy to observe tongue papillae, and to study the correlation between the condition of fungiform papillae and taste function. METHODS: Ten subjects (3 men, 7 women) were divided into two groups based on the results of a taste examination by electrogustometry. We compared the shape and blood vessels of the papillae between normal taste and taste disorder groups. RESULTS: In the normal taste group, round shaped papillae and clear blood vessels were observed with both microscopy and contact endoscopy. In the taste disorder group, flat and irregular papillae were observed with microscopy. Blood vessel flow of the papillae was observed to be poor with contact endoscopy. CONCLUSION: These findings suggested that the images of microscopy and contact endoscopy were related to taste function, and both techniques were useful for evaluating taste function.     

Long-term outcomes and complications associated with the use of a Groningen voice prosthesis

We have been using the Groningen voice prosthesis as a method of voice restoration after total laryngectomy for approximately five years. During this period, the Groningen voice prosthesis has been used in 19 patients and a total of 125 unit replacements have been performed. No serious complications have occurred to date. Upon examination of the voice restoration results, exchange frequency, and complications, we noted that 15 out of 19 patients (78.9%) were able to maintain a good voice quality, including cases with long-term observation periods. Voice restoration was difficult in the remaining 4 cases. The overall mean exchange period was 4.5 months, with 2.5 months being the shortest exchange interval in cases without complications. The mean exchange period for cases without complications was 6.1 months. No serious complications, such as a foreign body in the trachea, were encountered. However, several problems with water leakage occurred and were managed appropriately. Aspiration pneumonia from repeated water leakage did not occur, and no cases of TE shunt closure were encountered. These problems may occur with further aging. Thorough follow-ups will be continued in the future.     

HIV-1 Tat inhibits long-term potentiation and attenuates spatial learning [corrected

Although memory deficits have been clearly documented in patients with human immunodeficiency virus type-1 (HIV-1) infection, the physiological basis of this dysfunction is poorly understood. We focused on Tat, a viral protein released from HIV-1-infected cells and investigated its effect on spatial learning in adult mice. An intracerebroventricular injection of Tat leads to attenuation of spatial learning accompanied by suppression of long-term potentiation (LTP), the cellular basis of spatial learning, in hippocampal cornu ammonis 1 pyramidal neurons. Tat facilitates extrasynaptic but not synaptic N-methyl-D-aspartate (NMDA) receptor activity. Taken together, these data provide strong evidence that the Tat pathway underlies the development of memory dysfunction in patients with HIV-1 infection and suggest a causal relationship between Tat, the facilitation of extrasynaptic NMDA receptor activity, inhibition of LTP, and attenuation of spatial learning.     

Compartmentation of the mouse cerebellar cortex by sphingosine kinase

Classic cerebellar anatomy is based on the characteristic array of lobes and lobules. However, there is substantial evidence to suggest that more fundamental architecture is built around arrays of parasagittal stripes, which encompass both the inputs and outputs of the Purkinje cells (PCs). Sphingosine kinase (SPHK) is an enzyme that converts sphingosine (Sph) into sphingosine-1-phosphate (S1P). Recent reports have indicated that ceramide, Sph, and S1P play a role in cell survival, growth, and differentiation in several cell types, including neurons. In this study, we examined the localization of SPHK in the mouse cerebellum by using immunohistochemistry. Anti-SPHK immunoreactivity appeared in the cerebellar molecular layer and the PC membranes. The staining pattern is striped. In the molecular layer, the staining pattern probably reflects dendritic spines and dendrites. By electron microscopy, peroxidase reaction product was deposited within dendrites especially along the plasma membranes near spines. Seen at higher magnification, the staining was in and near the postsynaptic complexes. By double immunostaining, the striped pattern of SPHK expression was shown to be identical to that revealed by anti-zebrin II, although the subcellular distribution within PC's is not. This is the first demonstration of the cerebellar compartmentation of an enzyme related to lipid metabolism, and as such, it provides an insight into the roles of SPHK and formation of S1P. The selective expression of SPHK in the zebrin II-immunoreactive PCs may explain their resistance to cell death when ceramide metabolism is disrupted, as in the acid sphingomyelinase knockout model of Niemann-Pick type A/B disease.