Trehalose embedding technique for high-resolution electron crystallography: application to structural study on bacteriorhodopsin

Trehalose was compared with glucose in its use as an embedding medium for the preparation of two-dimensional crystals for electron crystallographic structure analysis. Purple membranes (two-dimensional crystals of bacteriorhodopsin) embedded in either sugar were studied in a 'partially hydrated' state as well as after 'drying' of the specimen. Diffraction patterns of the four conditions, all showing spots to a resolution better than 3.0 A, were analysed and merged. In the case of glucose embedded specimens, the merging R-factor obtained from the diffraction patterns of dried specimens was significantly better than the one obtained from partially hydrated specimens. In the case of trehalose embedding, the merging R-factor for partially hydrated specimens was significantly better than the dried one. The trehalose embedded, partially hydrated specimens gave the best merging R-factor in all four cases.     

Regulation of selectin binding activity by cyclization of sialic acid moiety of carbohydrate ligands on human leukocytes

We provide here evidence that supports the occurrence of a biologically dormant form of selectin ligand carbohydrate, the sialyl 6-sulfo Lewis X containing modified sialic acid, in human leukocytes. The modification of sialic acid involves first de-N-acetylation of sialic acid moiety through ubiquitous de-N-acetylation/re-N-acetylation cycle, followed by the dehydrative cyclization of de-N-acetyl sialic acid to form "cyclic sialic acid." The enzyme involved in the dehydration of de-N-acetyl sialic acid is a calcium-dependent enzyme having neutral-alkaline pH optimum. De-N-acetyl sialyl 6-sulfo Lewis X retained selectin binding activity as well as parental sialyl 6-sulfo Lewis X, but cyclic sialyl 6-sulfo Lewis X was devoid of selectin binding activity. Sialyl 6-sulfo Lewis X carrying the cyclic sialic acid is specifically recognized by the newly generated mAb, G159. The determinant was distributed widely among normal human leukocytes, especially on monocytes and subsets of lymphocytes including NK cells, helper memory T cells, Tcr-gammadelta T cells, and a part of B cells. The determinant was detected also on several cultured lymphocytic leukemia cell lines and O-tetradecanoylphorbol 13-acetate-activated lymphoid cells. Cyclic sialyl 6-sulfo Lewis X is efficiently formed by the action of the partly membrane-bound calcium-dependent enzyme, tentatively called "sialic acid cyclase," and a possible physiological significance of this reaction could be a rapid inactivation of selectin binding activity at the cell surface. Conversely, the accumulated intracellular cyclic sialyl 6-sulfo Lewis X determinant may function as a dormant pool of selectin ligands, which, on appropriate stimulation, is hydrolyzed and becomes active in selectin-dependent cell adhesion.     

Effects of the presence of the mammary gland on expression of neutrophil adhesion molecules and myeloperoxidase activity in periparturient dairy cows

Neutrophil function is reduced in periparturient dairy cows. Possible factors that reduce neutrophil function include endocrine changes associated with parturition and metabolic stresses associated with lactogenesis. In this study, mastectomized and intact cows were studied to specifically examine the effects of lactogenesis on neutrophil function in periparturient cows. Expression of adhesion molecules on neutrophils (L-selectin, mediating capture and rolling adhesion, and beta 2-integrin, mediating tight adhesion vital to egress) and neutrophil myeloperoxidase activity (an index of bactericidal activity) were assessed in mastectomized and intact cows. Expression of L-selectin decreased at parturition followed by rapid recovery to prepartum values in both intact and mastectomized cows. Expression of beta 2-integrins increased in intact cows at parturition but not in mastectomized cows. Expression of beta 2-integrins was greater in intact cows than in mastectomized cows throughout the study. Neutrophil myeloperoxidase activity decreased from baseline prepartum values as parturition approached in both intact and mastectomized cows, which suggests the endocrine changes associated with the act of parturition are predominant factors causing loss of neutrophil function. Myeloperoxidase activity recovered to prepartum values within a week of parturition in mastectomized cows; however, myeloperoxidase activity remained depressed in neutrophils obtained from intact cows throughout the first 20 d of lactation. The presence of the mammary gland and its attendant metabolic stresses slowed recovery of neutrophil function, which suggests that the metabolic stress of lactation exacerbated periparturient immunosuppression.     

Purification and some properties of a chitinase from Xanthomonas sp. strain AK

Chitinase B (ChiB) was purified from the culture supernatant of Xanthomonas sp. strain AK by Phenyl-Toyopearl 650M and DEAE-Toyopearl 650M column chromatographies. The purified enzyme preparation gave a single band on SDS-polyacrylamide gel electrophoresis and the molecular weight of ChiB was estimated to be 48,000. The enzyme was optimally active at pH 6.0 and 60 degrees C. N-Terminal amino acid sequence analysis suggested that ChiB is a member of glycosyl hydrolase family 18 and that it is genetically different from ChiA recently reported (Sakka et al., J. Ferment. Bioeng., 86, 527-533, 1998). Immunological analysis suggested that ChiB was the major chitinase species in the culture supernatant of Xanthomonas sp. strain AK and that production of the enzyme was induced by the presence of chitin.     

m-Calpain (calcium-activated neutral proteinase) in Alzheimer''s disease brains

The possible association between the emergence of cytopathogenic HIV-1 variants and disturbance of the cytokine production in the course of HIV-1 infection was studied in 18 infected patients. The cytopathogenicity of the isolates was studied in a microassay based on the use of HIV-1-infectible Hela-CD4 cells carrying the bacterial LacZ gene under the control of the HIV-LTR (P4 cells). In addition, the production of cytokines by heparinized whole blood (HWB) obtained the same day from HIV-1(+) patients was measured. TNF-alpha was determined in a one-step procedure combining HWB culture in the presence of LPS+PHA for 24 h and detection of cytokines in the same wells. In separate experiments HWB was cultured in the presence of LPS+PHA for 48 h, then the supernatants were collected and stored until assayed by ELISa for IFN-gamma and IL-4. Higher TNF-alpha levels were found in activated HWB of patients with cytopathic strains (n = 9) than in patients with non-cytopathic strains (n = 9, p = 0.02) assessed with P4 cells. A defective production of type 1 cytokine (IFN-gamma) and no increased secretion of type 2 cytokines (IL-4) was observed in patients with cytopathic strains. IFN-gamma/IL-4 ratios were significantly lower in patients with cytopathic strains (n = 9) than in other patients (n = 9, p = 0.009). The results show that the disarray of cytokine production, as assessed with whole blood culture, is associated with the cytopathogenicity of HIV-1 isolates in HIV-1-infected individuals.     

Elimination of artifacts in MRCP: technical consideration

Artifacts of MR cholangiopancreatography (MRCP) include fluid-filled gastrointestinal tracts, respiratory motion artifacts, spasm of Oddi's sphincter, vascular compression of bile ducts, overlapping of various anatomical structures and bright signal of surrounding fatty tissue. In this article, various technical considerations to eliminate these artifacts were described for the better imaging analysis of MRCP. The use of high-concentration ferric ammonium citrate (Ferriseltz) is recommended to reduce bright signal of fluid-filled gastrointestinal tracts. In case of long breathhold sequences, O2-inhalation study is useful to eliminate respiratory motion artifacts. Careful attention should be paid to the spasm of Oddi's sphincter and the vascular compression of bile ducts to avoid erroneous interpretation of MRCP findings.     

Chemotherapy of disseminated germ cell tumors

Current modes and issues of chemotherapy for the patients with disseminated germ cell tumors (GCTs) are described. A review of the literature of prospective randomized trials designed to compare the efficacy and toxicities between induction chemotherapy regimens for patients with either good- or poor-risk disseminated GCTs showed that three cycles of bleomycin, etoposide, and cisplatin (BEP) or four cycles of etoposide and cisplatin (EP) for good-risk GCTs, and four cycles of BEP for poor-risk GCTs, are the most effective regimens even now. A prognostic factor-based staging system can distinguish patients with either good- or poor-risk GCTs, and help make risk-based decisions about therapeutic modes. However, there is a variety of criteria that make intertrial comparison difficult. An internationally accepted prognostic classification of disseminated GCTs reported recently by the International Germ Cell Cancer Collaborative Group will resolve these problems. High-dose chemotherapy (HDCT) with autologous stem cell rescue can cure a relatively small but significant percentage of heavily pretreated patients who are deemed incurable with any other therapeutic strategy. Moreover, HDCT in first-line therapy for patients with poor-risk GCTs is now expected to improve treatment outcome obtained by BEP. Because HDCT has not totally overcome cisplatin-resistance, further investigation should be required.     

Dopamine receptor-mediated mechanisms involved in the expression of learned activity of primate striatal neurons

To understand the mechanisms by which basal ganglia neurons express acquired activities during and after behavioral learning, selective dopamine (DA) receptor antagonists were applied while recording the activity of striatal neurons in monkeys performing behavioral tasks. In experiment 1, a monkey was trained to associate a click sound with a drop of reward water. DA receptor antagonists were administered by micropressure using a stainless steel injection cannula (300 microm ID) through which a Teflon-coated tungsten wire for recording neuronal activity had been threaded. Responses to sound by tonically active neurons (TANs), a class of neurons in the primate striatum, were recorded through a tungsten wire electrode during the application of either D1- or D2-class DA receptor antagonists (total volume <1 microl, at a rate of 1 microl/5-10 min). Application of the D2-class antagonist, (-)-sulpiride (20 micrograms/microl, 58 mM, pH 6.8), abolished the responses of four of five TANs examined. In another five TANs, neither the D2-class antagonist nor the D1-class antagonists, SCH23390 (10 micrograms/microl, 31 mM, pH 5.7) or cis-flupenthixol (30 micrograms/microl, 59 mM, pH 6.6) significantly suppressed responses. In experiment 2, four- or five-barreled glass microelectrodes were inserted into the striatum. The central barrel was used for extracellular recording of activity of TANs. Each DA receptor antagonist was iontophoretically applied through one of the surrounding barrels. SCH23390 (10 mM, pH 4.5) and (-)-sulpiride (10 mM, pH 4.5) were used. The effects of iontophoresis of both D1- and D2-class antagonists were examined in 40 TANs. Of 40 TANs from which recordings were made, responses were suppressed exclusively by the D2-class antagonist in 19 TANs, exclusively by the D1-class antagonist in 3 TANs, and by both D1- and D2-class antagonists in 7 TANs. When 0.9% NaCl, saline, was applied by pressure (<1 microl) or by iontophoresis (<30 nA) as a control, neither the background discharge rates nor the responses of TANs were significantly influenced. Background discharge rate of TANs was also not affected by D1- or D2-class antagonists applied by either micropressure injection or iontophoresis. It was concluded that the nigrostriatal DA system enables TANs to express learned activity primarily through D2-class and partly through D1-class receptor-mediated mechanisms in the striatum.     

Nucleotide sequencing analysis of the 146-kilobase segment around the IkBL and MICA genes at the centromeric end of the HLA class I region

The biomechanics of medialization laryngoplasty are not well understood. An excised canine larynx model was used to test the effects of various sized silicon implants. The vocal fold length, position, and tension were measured. Medialization laryngoplasty did not affect vocal fold length. At the mid-membranous vocal fold, larger shims resulted in greater medialization and tension. Medialization laryngoplasty neither medialized nor stiffened the vocal process to resist lateralizing forces. We conclude that medialization laryngoplasty provides bulk and support for defects of the membranous region of the vocal fold, but does not appear to close a posterior glottal gap. The selection of a surgical procedure to treat glottal incompetence should take into account the unique biomechanical properties of the anterior (membranous vocal folds) and posterior (cartilaginous portion) glottis.