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.     

Effect of capsulectomy on the hemodynamics and viability of random-pattern skin flaps raised on expanded skin in the pig

Skin flaps constructed on expanded skin usually include the underlying capsular tissue. It has been hypothesized that capsulectomy may jeopardize the viability of the expanded skin flap. The experiments reported herein were designed to test this hypothesis. Specifically, we studied the hemodynamics and viability of random-pattern skin flaps (8 X 20 cm) raised on delayed bipedicle flaps (group A) and on expanded skin pockets with capsulectomy at the time of flap elevation (group B) or with intact underlying capsular tissue (group C). Each group was randomly assigned to each flank in 16 pigs. Skin pockets were expanded by inflation of subcutaneous silicone tissue expanders with sterile saline (299 +/- 7 ml; X +/- SEM) over a period of 3 weeks. At the end of this period, the bipedicle flaps were constructed. Eight days later, random-pattern skin flaps were raised on bipedicle flaps and skin pockets. The length and area of skin flap viability, judged by the fluorescein dye test performed 1 day postoperatively, were not significantly different (p greater than 0.05) among groups A, B, and C (n = 31 to 32). There also were no significant differences (p greater than 0.05) in total skin capillary blood flow measured 1 day postoperatively (A = 2.6 +/- 0.4, B = 2.4 +/- 0.4, and C = 2.7 +/- 0.6 ml/min per flap; n = 15 to 16) and in skin viability assessed 7 days postoperatively (A = 74 +/- 2, B = 75 +/- 2, and C = 76 +/- 2 percent; n = 16) among delayed skin flaps and skin flaps raised on expanded skin pockets with or without capsulectomy. The results of this flap viability study were confirmed in 5 minipigs in a separate experiment. We conclude that capsulectomy did not have a detrimental effect on the hemodynamics and viability of random-pattern skin flaps raised on expanded skin. Furthermore, we hypothesize that skin flaps raised on expanded skin are similar to delayed skin flaps in that the skin blood flow is optimally augmented; therefore, the capsular tissue does not add significant blood supply to the overlying skin.     

Propranolol antagonizes hypotension induced by alpha-blockers but not by sodium nitroprusside or methacholine

A pressor response has been observed with propranolol, a nonselective beta-adrenoceptor antagonist, in animals given a nonselective alpha-adrenoceptor antagonist. This study investigates whether a pressor response to propranolol occurs in conscious unrestrained rats following a hypotensive response induced by phentolamine (nonselective alpha-antagonist), prazosin (selective alpha 1-antagonist) and (or) rauwolscine (selective alpha 2-antagonist), sodium nitroprusside (smooth muscle relaxant), or methacholine (muscarinic agonist). The rats were subjected to a continuous infusion of a hypotensive agent or normal saline followed by i.v. injection of propranolol. The infusion of phentolamine significantly decreased mean arterial pressure (MAP). Subsequent injection of propranolol restored MAP to the control level. Prazosin and rauwolscine each caused a small but not significant decrease in MAP which was reversed by propranolol. Concurrent infusions of prazosin and rauwolscine caused a significant decrease in MAP. Subsequent injection of propranolol caused a large pressor response which increased MAP to 20% above control MAP prior to the administration of drugs. Nitroprusside or methacholine each caused a significant decrease in MAP, but the hypotension was not antagonized by propranolol. The concurrent infusions of a low dose of nitroprusside and prazosin caused a significant decrease in MAP which was reversed by propranolol. The infusion of saline did not alter MAP, and propranolol did not cause a pressor response. It is concluded that propranolol antagonizes the hypotensive effect of an alpha-blocker but not that of sodium nitroprusside or methacholine. Our results suggest the presence of a specific interaction between alpha- and beta-antagonists.     

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.     

Evidence that insulin receptor from human placenta has a high affinity for only one molecule of insulin

Insulin receptor, partially purified from human placenta by chromatography on wheat germ agglutinin, was shown, by means of double probe labeling, to bind only one molecule of insulin with a high affinity. In the double probe labeling protocol used, 125I-insulin (probe 1) was affinity cross-linked to its receptor in the presence of an excess of unlabeled N epsilon B29-biotinylinsulin (probe 2). The ability of succinylavidin to bind to receptor-linked probe 2 and alter the electrophoretic mobility of the cross-linked complex (during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) was used to determine the amount of receptor which was cross-linked to both probes relative to that which was cross-linked to only probe 1. The fraction of receptor bound to two molecules of insulin prior to cross-linking was estimated from the cross-linking efficiency and the yield of receptor cross-linked to both probes relative to the yield of receptor cross-linked only to probe 1. The low fraction of receptor bound to both probes in the presence of high concentrations of probe 2 indicated that the affinity of the receptor for a second molecule of insulin was approximately 100 times less than that for the first and that in the range of insulin concentrations (less than 20 nM) usually used to determine the stoichiometry for the interaction between receptor and insulin, more than 80% of the receptor molecules should be bound to only one molecule of insulin. This knowledge of how insulin receptor interacts with insulin was shown to be important for proper determination of receptor purity, interpretation of curvilinear Scatchard plots, and interpretation of the insulin-enhanced rate of dissociation of receptor-bound insulin.     

广西德峨苗族、彝族体质调查

对广西隆林县德峨乡男性22至60岁与女性20至60岁的576名苗族人(男395、女181)和178名彝族人(男88、女90)进行了活体观察与测量,计算出各项数据,总结德峨苗族、彝族的体质特征,并与国内一些民族相比较。     

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.     

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.