Addition of Cryoprotectant Significantly Alters the Epididymal Sperm Proteome

Although cryopreservation has been developed and optimized over the past decades, it causes various stresses, including cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. During cryopreservation, addition of cryoprotective agent (CPA) is crucial for protecting spermatozoa from freezing damage. However, the intrinsic toxicity and osmotic stress induced by CPA cause damage to spermatozoa. To identify the effects of CPA addition during cryopreservation, we assessed the motility (%), motion kinematics, capacitation status, and viability of epididymal spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, the effects of CPA addition were also demonstrated at the proteome level using two-dimensional electrophoresis. Our results demonstrated that CPA addition significantly reduced sperm motility (%), curvilinear velocity, viability (%), and non-capacitated spermatozoa, whereas straightness and acrosome-reacted spermatozoa increased significantly (p < 0.05). Ten proteins were differentially expressed (two decreased and eight increased) (>3 fold, p < 0.05) after CPA, whereas NADH dehydrogenase flavoprotein 2, f-actin-capping protein subunit beta, superoxide dismutase 2, and outer dense fiber protein 2 were associated with several important signaling pathways (p < 0.05). The present study provides a mechanistic basis for specific cryostresses and potential markers of CPA-induced stress. Therefore, these might provide information about the development of safe biomaterials for cryopreservation and basic ground for sperm cryopreservation.     

Base-catalyzed oligomerization of levoglucosenone

Heating levoglucosenone in aqueous triethylamine gives a dimer and two trimers in yields of 8, 18, and 56%, respectively. These compounds have been isolated crystalline, and their structures and stereochemistry have been investigated by ¹³C- and ¹H-n.m.r. and other spectroscopic methods. These data indicate that the dimer is apparently formed by Michael addition to provide a C-3-C-4 linkage. Similar reactions provide a non-olefinic, C-3-C-4-linked, cyclic trimer and an olefinic, cyclic trimer containing two C-3-C-4 linkages and one C-2-C-3 linkage.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Age-related changes in chemical composition and physical properties of mucus glycoproteins from rat small intestine.

Mucus glycoproteins from newborn and adult rat small intestine were radiolabelled in vivo with Na2 35SO4 and isolated from mucosal homogenates by using Sepharose 4B column chromatography followed by CsCl-density-gradient centrifugation. Non-covalently bound proteins, lipids and nucleic acids were not detected in the purified glycoproteins. Amino acid, carbohydrate and sulphate compositions were similar to chemical compositions reported for other intestinal mucus glycoproteins, as were sedimentation properties. There were, however, important differences in the chemical and physical characteristics of the mucus glycoproteins from newborn and adult animals. The buoyant density in CsCl was higher for the glycoproteins from newborn rats (1.55 g/ml versus 1.47 g/ml). On sodium dodecyl sulphate/polyacrylamide/agarose-gel electrophoresis, the glycoprotein from newborn rats had a greater mobility than the adult-rat sample. Although both preparations had similar general amino acid compositions, variations were observed for individual amino acids. The total protein content was greater in the glycoprotein from newborn animals (27%, w/w, versus 18%, w/w). The molar ratio of carbohydrate to protein was less in the newborn, primarily owing to a decreased fucose and N-acetylgalactosamine content. Comparison of the molar ratio of fucose and sialic acid to galactose for both glycoproteins demonstrated a reciprocal relationship similar to that described by Dische [(1963) Ann. N.Y. Acad. Sci. 106, 259-270]. The sulphate content was greater in the glycoprotein from newborn rats (5.5%, w/w, versus 0.9%, w/w). Both had similar sedimentation coefficients in a dissociative solvent. These results suggest an age-related difference in the types of mucus glycoproteins synthesized by small intestine.     

泥炭藓群落的光谱特征及遥感识别研究

以中位泥炭藓（Sphagnum magellanicum Brid.）为研究对象,分别从实测冠层光谱和遥感传感器模拟光谱层面分析其群落的光谱特征。研究结果显示,中位泥炭藓与北方针叶林光谱差异明显,最佳光谱识别区间为740~1140 nm和1230~1412 nm。在可见光波段上,中位泥炭藓与云杉（Picea engelmannii Parry ex Engelmann）和黑松（Pinus contorta Douglas ex Loudon）的绿峰位置有所差异。水竹（Phyllostachys heteroclada Oliver）和中位泥炭藓的光谱识别特征波段集中在可见光-近红外波段,分别为400~550、560~696、1025~1143 nm。中位泥炭藓与北方针叶林以及水竹的特征光谱区间存在细微差异,且与水竹在可见光波段有较好的可分性,因此不同纬度带上中位泥炭藓群落的特征谱宽有所差异。红外波段是中位泥炭藓识别的最佳光谱区间。在多光谱遥感水平上,中位泥炭藓识别效果较好,传感器的识别能力依次为：MSI > ALI > OLI > ASTER。在2个中位泥炭藓群落的光谱特征分析中,导数、对数、包络线去除法的光谱降维能力有所差异,其中包络线去除法效果最好。     

Optimization of microalgal production in a shallow outdoor flume

The marine diatom Cyclotella cryptica was grown over a period of 13 months in a 48-m(2) shallow outdoor flume. The use of foil arrays at intervals of 1.2 m to effect systematic vertical mixing in the flume was found to significantly enhance microalgal production (p = 0.006). Average photosynthetic efficiencies (based on visible irradiance) with and without the foil arrays in place were 9.6 +/- 0.8 and 7.5 +/- 0.5% (+/-95% confidence intervals), respectively. A cost-benefit analysis indicated that the foil arrays were cost-effective if the value of the algae exceeded about $2.28 kg(1) of ash-free dry weight (AFDW). Parallel experiments performed in four 9.2-m(2) flumes showed that production was maximized when the cells were grown on a 2-day batch cycle between harvests rather than on a 1- or 3-day batch cycle. The optimum initial concentration (immediately after harvesting) of the algae was negatively correlated with the time interval between harvests and ranged from approximately 39 g AFDW/m(3) on a 3-day cycle to 213 g AFDW/m(3) on a 1-day cycle. The increase in production resulting from growth on a 2-day rather than a 1-day batch cycle was about 19% and was statistically significant at p = 0.0003. Growth of C. cryptica over a total period of 122 days during the 13-month study in the 48-m(2) flume under near-optimal conditions (2-day batch cycle, initial concentration 155 g AFDW/m(3)) resulted in an average production rate (+/-95% confidence interval) of 29.7 +/- 2.7 g AFDW/m(2) d.     

Augmentation of blood flow in delayed random skin flaps in the pig: effect of length of delay period and angiogenesis

Skin capillary blood flow and angiogenesis were studied by radioactive microsphere and morphometry technique, respectively, in delayed random skin flaps in the pig. Skin flaps were delayed for 2, 3, 4, 6, or 14 days. Blood flow was measured 6 hours after complete raising of acute and delayed random skin flaps on the opposite flanks of the same pig. It was observed that the capillary blood flow increased significantly (p less than 0.05) within 2 days of delay compared to the acute skin flaps. This capillary blood flow further increased by about 100 percent between days 2 and 3, started to plateau after day 3, and remained unchanged between days 4 and 14 of delay. This increase in capillary blood flow was mainly in the distal portion of the delayed skin flaps. There was no indication of an increase in the density of arteries in all delay periods studied. Our observations did not support the hypotheses that the delay phenomenon involves angiogenesis or long-term adaptation to ischemia, as have been hypothesized previously. The possible mechanism of delay is discussed.     

Hemodynamics and vascular sensitivity to circulating norepinephrine in normal skin and delayed and acute random skin flaps in the pig

Cutaneous circulation in 4 X 10 cm skin samples and delayed and acute random skin flaps constructed on the flanks of castrated Yorkshire pigs (13.3 +/- 0.7 kg; n = 12) were studied during intravenous infusion (0.5 ml per minute) of 5% dextrose solution (vehicle) and 5% dextrose containing norepinephrine (1 microgram/kg per minute). Total and capillary blood flow and A-V shunt flow were measured by the radioactive microsphere technique 6 hours after the raising of 4 X 10 cm single-pedicle acute and delayed random skin flaps using the technique and calculations published previously. Fluorescein dye test was also performed to assess vascular perfusion. It was observed that the capillary blood flow in the single-pedicle delayed skin flaps was similar to that in the normal skin, and the maintenance of this normal skin blood flow was not due to the closing of A-V shunt flow in the delayed skin flaps. Similarly, the significant (p less than 0.01) decrease in capillary blood flow and distal perfusion in the acute skin flaps compared with the delayed skin flaps was not due to the opening of A-V shunts in the acute skin flaps. There was no evidence to indicate that A-V shunt flow per se was the primary factor for the regulation of capillary blood flow in the acute and delayed skin flaps in the pig. Our data seemed to indicate that tissue ischemia in the distal portion of acute skin flaps was likely the result of vasoconstriction of the small random arteries which supplied blood to arterioles and A-V shunts, and locally released neurohumoral substances may play an important role in the pathogenesis of vascular resistance and ischemia in the acute skin flaps.     

Exfoliative cytology in the evaluation of interferon treatment of cervical intraepithelial neoplasia

Local interferon injection in four patients with cervical intraepithelial neoplasia (CIN) regularly elicited progressive regression of the lesions. The response was observed with exfoliative cytology after each injection, guided by colposcopic examination. The cytologic changes showed a cytocidal effect mainly on the dyskaryotic cells, preceded by cellular degeneration not unlike that of nonspecific inflammation and accompanied by an increase in neutrophil infiltration. The cytologic response was closely correlated with partial or complete clinical regression based on the absence of viable or degenerated dyskaryotic cells in the cervical smears. Three patients showed complete clinical regression after treatment. One patient showed recurrent viable dyskaryotic cells when the dosage was reduced, and treatment was suspended temporarily although her lesion had regressed completely after five injections. Clinical recurrence was noted one week after viable dyskaryotic cells reappeared in her smears. These observations suggest that cytology may be a useful means of monitoring interferon treatment in CIN.     

New view of the surface projections of Chlamydia trachomatis.

Two kinds of surface specializations of chlamydiae have been described: hemispheric projections and spikelike rods. We undertook the present studies to demonstrate chlamydial ultrastructure in greater detail in conventional thin-sectioned specimens. Chlamydia trachomatis (LGV strain L2/434/Bu), cultured for 40 h in L929 mouse fibroblasts, was fixed in glutaraldehyde-acrolein, p-formaldehyde-glutaraldehyde, or glutaraldehyde-osmium tetroxide mixtures, postfixed in osmium tetroxide, stained in uranyl acetate, dehydrated in ethanols, and embedded in Epon. By the use of fixatives that penetrate and fix rapidly, chlamydial outer and plasma membranes were clearly revealed. Our results indicate that the hemispheric projections are specializations of the plasma membrane of elementary bodies. The spikelike projections are found in intermediate forms, originate beneath depressions of the plasma membrane, and extend through the periplasmic space and outer membrane to end with pointed tips. Improved preservation of chlamydiae provides a new, informative view of their complex structure. Significant interactions between chlamydiae and host cells might be influenced by the surface structures shown in this study.