Studies of the fossil dinosaur bone in the scanning electron microscope.

A fossil dinosaur bone, 80 million years old, was subjected to investigation in the scanning microscope. The bone surfaces to be examined were prepared with appropritely modified methods used in the technique of replication in transmission electron microscopy. In the scanning microscope pictures of vascular canals were obtained. The walls of these canals were shown to be formed of collagen fibrils. Moreover, it was demonstrated that the internal surface of the canal wall is made up of bundles of collagen fibrils which run obliquely, corkscrewwise, and in the form of plexus to the long axis of tke canal; Besides, osteocytes of the dinosaur bone were isolated and pictures of their spatial structure together with characteristic points of departure of processes from the cell body were obtained.     

Spatial organization of the lacunar-canalicular system in the structures of bone lamellae

By means of light microscopical techniques and scanning electron microscopy spatial organization of the lacunar-canalicular system (LCS) has been studied in structures of a mature lamellar bone. A method for making corrosive casts of osseous lacunae and canaliculi is suggested, owing to which their spatial organization can be analysed. Certain data on interconnections of the osseous lacunae with each other and with vascular canals and natural surfaces of the bone are presented. The role of LCS as a component of the microcirculatory bed of the lamellar bone is discussed.     

Ruthenium Red-Mediated Osmium Binding for Examining Uncoated Biological Material Under the Scanning Electron Microscope

A simple technique for examining uncoated soft biological material under the scanning electron microscope is described. Rat tissues were initially fixed in 2.5% glutaraldehyde either by intravascular perfusion or by immersion. The samples were placed in buffered 2.5% glutaraldehyde containing 0.05% ruthenium red and postfixed in buffered 1% osmium tetroxide containing 0.05% ruthenium red. The samples were alternately incubated 3 to 5 times in 1% O₃O₄ and 0.1% ruthenium red solutions with continuous shaking at room temperature. The specimens were dehydrated, critical point dried, mounted and examined under the scanning electron microscope. Contour details were clearly defined at both the external and cut surfaces of the tissues. The specimens could be observed for more than 30 minutes without excessive charging or glow effects and the material remained stable under the beam at 20-25 kV and at various magnifications.     

Electron and light microscopy of neutrophil responses in mice vaccinated and challenged with third-stage infective hookworm (Ancylostoma caninum) larvae.

The role of neutrophils in mediating host inflammation was examined in mice vaccinated with living third-stage infective hookworm larvae (L₃). Mice were vaccinated by oral immunization with 500 L₃ (Ancylostoma caninum) once every 2 weeks for a total of three immunizations. The vaccinated mice were then challenged intraperitoneally with 2000 L_3, 1 week after the final immunization. To stimulate peritoneal production of neutrophils, 2 ml of 2% glycogen were injected intraperitoneally at 16 h prior to the challenge infection. Neutrophils were found to comprise 85% of the peritoneal cell population. L₃ from the challenge infection were collected and then examined at timed intervals by inverted light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Greater than a fivefold increase in the total numbers of peritoneal cells was noted in the vaccinated mice as compared to unvaccinated mice. In the peritoneal cavity of vaccinated mice, the neutrophils adhered to the L₃ within 2 h, and over 55% of the L₃ were surround by clusters of neutrophils to form a sausage-like sheath 4 h later. At 24–72 h after challenge, almost all of the L₃ recovered from the vaccinated mice were covered with thick clusters of cells. Both SEM and TEM demonstrated extensive ultrastructural damage to the L_3. In contrast, the L₃ recovered from the unvaccinated mice appeared to be unaffected by neutrophils. These studies suggest that neutrophils, like macrophages, can have an important role as effector cells in L₃-vaccinated mice.     

Use of a Tissue Sectioner to Expose Internal Structures of Biological Samples for Scanning Electron Microscopy

A procedure yielding sections of unembedded biological samples for observation by scanning electron microscopy is described. Sections of samples, fixed and hardened in OsO₄, were obtained in quantity with a tissue sectioner. Subsequent treatments to osmium-coat cut surfaces were employed prior to critical point drying. The procedure yields cleanly cut surfaces through cells and cytoplasmic organelles which are retained in their normal position. Sections of apple leaf and mouse kidney are illustrated. Sections can be readily cut in a desired plane with less structural damage than is typically encountered by other sectioning or dissection techniques.     

Straight Oso4 versus Glutaraldehyde-Oso4 In Sequence as Fixatives for the Granular Vesicles in Sympathetic Axons of the Rat Pineal Body

Pineal bodies were removed immediately after death from 6 rats: representing both sexes, and adult and 21-day postnatal ages; cut into 2 or 3 pieces, and subjected to experimental fixations at pH 7.3, 0-4 C as follows: 1-2 hr in 1% OsO₄, with veronal-acetate buffer of phosphate buffer; 3-4 hr in 3% or 6% glutaraldehyde in 0.1 M or 0.2 M phosphate buffer, with or without 1% sucrose. Specimens from OsO₄ were dehydrated, and embedded in epoxy resin; those from glutaraldehyde were allowed to soak in buffer for 12-16 hr, then transferred to 1% OsO₄ at 0-4 C for 2 hr, and embedded in the same manner as the ones fixed directly in OsO₄. Representative electron micrographs of postganglionic sympathetic endings were studied for the morphology and frequency of granular vesicles. No consistent difference was shown between vesicles fixed in OsO₄ buffered by phosphate or by veronal-acetate, nor was there any effect caused by the different concentrations used for the glutaraldehyde solution; however, vesicles fixed by the glutaraldehyde-OsO₄ sequence showed an enhancement in the graininess of their membranes, were slightly larger, and had a much larger dense core than those fixed by OsO₄ alone. After glutaraldehyde-OsO₄, granular vesicles showed a frequency of 81%, whereas after direct fixation in OsO₄, only 40% without significant change their number per unit area. Therefore, glutaraldehyde-OsO₄ seems to be more effective than straight OsO₄ for the demonstration of granular vesicles in the autonomic nervous system.     

Affinity separation of immunoglobulin G subclasses on dye attached poly(hydroxypropyl methacrylate) beads

Poly(hydroxypropyl methacrylate) [poly(HPMA)] gel beads with an average size of 150–200 μm were prepared by suspension polymerization of hydroxypropyl methacrylate (HPMA). The poly(HPMA) gel beads were characterized by swelling studies, surface area measurements, scanning electron microscopy (SEM) and elemental analysis. Poly(HPMA) gel beads had a specific surface area of 88.6 m²/g. The dye Reactive Green HE 4BD was chemically attached to yield dye-poly(HPMA) gel beads at an average concentration of 44.3 μmol dye/g bead with a swelling ratio of 75%. These dye attached gel beads were used in the separation of immunoglobulin-G (IgG) through adsorption–elution studies. The non-specific adsorption of IgG on the poly(HPMA) gel beads was 0.5 mg/g. The attachment of Reactive Green HE 4BD significantly increased the adsorption of IgG up to 71 mg/g. The Langmuir adsorption model was found to be applicable in interpretation of data pertaining to the adsorption studies of IgG with Reactive Green HE 4BD attached to the poly(HPMA) gel beads. The adsorption of IgG was found to be optimal at pH 7.0. The adsorption of IgG was observed to decrease by about 76% as the NaCl concentration was increased from 0.001 to 0.1 M. The IgG adsorption capacity of the dye attached poly(HPMA) gel beads was determined for a commercially available IgG solution to be 4.2 mg/g for IgG₁, 64.5 mg/g for IgG₂, 7.1 mg/g for IgG₃ and 10.8 mg/g for IgG₄. The Reactive Green HE 4BD attached poly(HPMA) gel beads have a significant adsorption capacity for IgG₂. The quantity of adsorbed IgG₂ is three times higher than the quantity of the other subclasses, IgG₁, IgG₃ and IgG₄. A similar adsorption behaviour was observed when the albumin free human plasma was used. The quantity of adsorbed IgG₂ is higher than the quantity of the other subclasses, IgG₁, IgG₃ and IgG₄. Adsorption capacities for albumin free human plasma were obtained as 6.4 mg/g for IgG₁, 67.8 mg/g for IgG₂, 5.2 mg/g for IgG₃ and 8.6 mg/g for IgG₄. Significant amount of the adsorbed IgG (up to 95%) was eluted in 1 h in the elution medium containing 2.0 M NaCl. Repeated adsorption/elution processes showed that these dye attached gel beads are suitable for IgG adsorption.     

An Infiltration and Clearing Technic to Reveal the Vascular Pattern of Ethylenediamine-Treated Cortical Bone

The vascular canals of cortical bone are revealed by: (1) removing all organic tissue with ethylenediamine (ed), (2) infiltrating the Haversian and Volkmann canals with a pigment and (3) finally clearing the specimens in styrene. Buccal and lingual cortical plates of mandibles were freed of spongy bone and placed in a Soxhlet extractor. Extraction was carried out with a solution consisting of 4 volumes of ed and 1 volume of distilled water which was maintained at a temperature of 118°C for 24 hr. After extraction, the organic-free specimens were washed in water for 1 hr and dried. The dried bone was immersed in a 25% India ink suspension and placed in a vacuum chamber until bubbling ceased. The bones were then dried and the surface carbon was removed with a CO₂-driven stream of dolomite powder from an Airdent unit. Compressed air was used to remove the residual dolomite powder from the specimens. They were then immersed in styrene until clear. The specimens could be studied directly from the styrene, since the inorganic portion of the bone became transparent and carbon retained on the walls of the vascular channels within the calcified tissue was clearly visible. Plastic can be substituted for styrene for infiltrating the spaces within the specimen which were previously occupied by organic material. After infiltration and curing of the plastic, the embedded specimens can be decalcified with concentrated HCl. After decalcification, the voids created by the removal of the inorganic matrix can then be filled with plastic. Substituting plastic for areas previously occupied by both the organic and inorganic elements results in an easily visualized, carbon-black three-dimensional preparation of the vascular pattern of bone.     

掺银二氧化钛空心球的制备及其抗菌性能研究

以硫酸钛、尿素为原料,碳球为模板,采用水热沉淀法制备了二氧化钛空心球,以聚乙烯吡咯烷酮(PVP)为分散剂、硼氢化钾还原硝酸银的方法得到银掺杂的二氧化钛空心球。采用X射线粉末衍射仪(XRD)、扫描电镜(SEM)、透射电镜(TEM)、能谱仪(EDS)和傅里叶变换红外光谱仪(FT-IR)等测试手段对所得的样品进行了表征。采用抑菌圈法对其抗菌性能进行了检测,实验结果表明,在自然光条件下,商品二氧化钛P25和二氧化钛空心球对大肠杆菌、金黄色葡萄球菌、白色念珠菌没有任何抗菌效果,而载银量9.4 mol%的二氧化钛在常温范围内对三个菌种都具有优良的抗菌性能,可用于抗菌塑料、抗菌食品包装和抗菌纺织制品等领域。     

Effects of prostaglandins of the E-series on pulmonary and systemic circulations of newborn goats during normoxia and hypoxia

Effects of exogenous prostaglandins of the E-series on pulmonary and systemic circulations of newborn goats were investigated during normoxia and hypoxia. Pulmonary arterial infusion of prostaglandins E₁ and E₂ decreased pulmonary vascular resistance 20% and 14%, respectively, without systemic effects. Prostaglandin E₁ abolished the pulmonary pressor response to hypoxia. Prostaglandin E₂ was less effective in counteracting this hypoxic response. The increased pulmonary vascular resistance and augmented response to hypoxia following indomethacin administration was reversed by prostaglandin E₁. Infusion of prostaglandin E₁ directly into the pulmonary circulation may be of benefit to the distressed newborn with elevated pulmonary vascular resistance.     

The b-type cytochromes of bovine heart mitochondria: Absorption spectra, enzymatic properties, and distribution in the electron transfer complexes

1. 1. Three b-type cytochromes (b_557.5, b₅₆₀, and b_562.5), plus a chromophore with an absorption peak at 558 nm at 77 °K, have been found to be associated with the electron transport system of bovine heart mitochondria. The reduced minus oxidized spectra of these components at 77 °K, as well as that of cytochrome c₁, have been recorded with a wavelength accuracy of ± 0.1 nm and presented to the nearest 0.5 nm. All the major and β absorption peaks of cytochromes b_557.5, b₅₆₀, b_562.5, c₁ and c have been shown by fourth derivative analysis to be present in the dithionite-reduced minus oxidized spectra of mitochondria and submitochondrial particles.

2. 2. The distribution of the above components has been studied in the four electron transfer complexes of the respiratory chain. Cytochromes b₅₆₀, b_562.5 and c₁, as well as chromophore-558, were found to fractionate into Complex III (reduced ubiquinone-cytochrome c reductase), whereas cytochrome b_557.5 was found in Complex II (succinate-ubiquinone reductase).

3. 3. Cytochrome b₅₆₀ was readily reduced by NADH or succinate, but b_562.5 was not reduced by substrates unless the preparation was treated with antimycin A. In antimycin-treated preparations pre-reduction of c₁ with ascorbate inhibited the subsequent reduction of b_562.5 by substrates. These results indicate that b₅₆₀ and b_562.5 correspond, respectively, to b_K and b_T previously described by Chance et al.¹⁴ (1970, Proc. Natl. Acad. Sci. U.S. 66, 1175–1182).

4. 4. Similar to b₅₆₀, chromophore-558 can be reduced by substrates in the absence or presence of antimycin A. However, in antimycin-treated preparations, pre-reduction of c₁ inhibits its subsequent reduction by substrates. This property is similar to that of b_562.5.

5. 5. Cytochrome b_557.5, which occurs in Complex II, appears to have a low mid-point potential. It can be reduced with dithionite and oxidized by fumarate or ubiquinone. CO treatment of dithionite-reduced b_557.5 neither modified the spectrum of this cytochrome nor diminished the extent of b_557.5 reoxidation by fumarate.

6. 6. Antimycin A treatment does not appear to alter the spectra of the above cytochromes. However, small amounts (< 4%) of ethanol or methanol, which are usually added to particles as solvent for antimycin A, have a pronounced effect on the peaks of cytochrome c₁. The spectrum of cytochrome c₁ at 77 °K as modified by 3% (v/v) ethanol is shown.

Preparation and characterization of combi-CLEAs catalyzing multiple non-cascade reactions

A novel cross-linked enzyme aggregates (CLEA) concept called combi-CLEA has been described. It is based upon the fact that CLEA can be made from heterogeneous populations of proteins/enzymes. Porcine pancreatic acetone powder crude extract was used for preparing CLEA in such a way that lipase, -amylase, phospholipase A₂ activities were retained upto 100%. The lipase present in the CLEA showed greater thermal stability at 50 °C as compared to free enzyme. For lipase and phospholipase A₂, V_max/K_m showed no significant change upon combi-CLEA formation but decreased significantly for -amylase activity from 190 to 114 min⁻¹. The lipase activity and -amylase activity in CLEA were completely retained upto three cycles of use. The scanning electron microscopic (SEM) studies showed that morphology of CLEA changed upon inactivation by reuses.     

Changes in femoral morphology during egg-laying in Alligator mississippiensis

Birds and many reptiles are egg-layers. Birds provide calcium for the formation of eggshells by resorbing medullary bone, which is laid down before ovulation. Turtles do not possess this mechanism and resorb structural bone to form eggshells. Femora from three groups of alligators (egg-laying females; quiescent, immature, or barren females; and males) were examined to determine if alligators, which are closely related to birds in evolution, resorb structural bone during the formation of eggshells as do turtles. Microradiographs of cross sections from femoral mid-shafts were analyzed for porosity, and the robusticity index of each femur was determined. Scanning electron micrographs of anorganic endosteal and periosteal femoral surfaces were analyzed to determine numbers of entrances of vascular canals, numbers of lacunae of osteoblasts, and types of femoral surfaces. Femora from egg-laying females were significantly less robust than those of other females or males, and sections of bone from the egg-layers were significantly more porous than those of the other groups. Scanning electron microscopy of anorganic femoral endosteal surfaces from egg-laying females revealed significantly more resorption areas when compared with males or non egg-laying females. Periosteal surfaces from egg-layers had significantly more resting and less bone-forming surface than those from the other groups. Results indicated that apposition of periosteal bone may have been reduced in egg-layers and that egg-laying alligators, like turtles, resorb endosteal structural bone, which may be used as a source of calcium for the formation of eggshells.     

Studies on the enzyme systems involved in electron and energy transfer in isolated chloroplasts. I. Effect of endogenous phosphate on the photophosphorylation coupled with noncyclic electron transport in intact chloroplasts

1. It was found that the P/2e ratio was independent of the degree of electron transport stimulation in the presence of ADP and P_i and exceeded 1.0 in the preparations with slight (30 %) as well as with high (80 %) stimulation.

2. Chloroplast preparations having a low content of endogenous P_i showed higher stimulation than those with higher contents.

3. Illumination of the chloroplasts in the presence of ADP and electron acceptor led to a decrease of endogenous P_i content that resulted in an increase of electron transport stimulation in the presence of exogenous P_i.

4. Electron transport in the absence of exogenous P_i was inhibited by both exogenous ADP and ATP.

5. It appears that the electron transport in the absence of exogenous P_i is coupled to phosphorylation, which occurs because isolated chloroplasts contain endogenous P_i. Stimulation of the electron transport by the addition of ADP and P_i seems to be caused by acceleration of the existing electron transport pathway, and not from the initiation of a new one.     

Stress relaxation function of bone and bone collagen

Relaxation Young's and shear moduli of bovine bone and bone collagen were investigated. It was found that each relaxation process observed had two stages, which were referred to as process I and process II in order of time. Process II was described by a simple exponential decay while process I was not. The Kohlrausch-Williams-Watts (KWW) function, ψ(t) = exp[t/τ₁)^B] (0 < B < 1), was found to be suitable to describe process I. The normalized relaxation modulus, M_r(t), was expressed by the combination of the simple exponential type relaxation function and the KWW function

M_r=A₁exp[−(t/τ₁)^B]+A₂exp[(t/τ₁)](0<B1)

On the basis of this equation, the relaxation mechanism in bone and bone collagen was identified. According to the model proposed for the KWW relaxation function, the stress relaxation process in bone was considered to be governed by viscoelastic properties of matrix collagen fiber. The model for the KWW relaxation function requires the disordered glassy structure of collagen fiber, which is consistent with the results of the structural investigations.     

Electron transport between plastoquinone and chlorophyll aI in chloroplasts

After preillumination with System I light spinach chloroplasts were excited by one flash or a group of saturating flashes. During the following dark period the time courses of the oxidation of plastohydroquinone and of the simultaneous reduction of oxidized cytochrome f and chlorophyll a_I (P700) have been measured.

1. 1. From a correlation of these kinetics it can be concluded that at least 85% of the electrons from plastohydroquinone are transferred to chlorophyll a_I.

2. 2. After one flash 93% of the oxidized chlorophyll a_I is reduced. This suggests a high equilibrium constant between chlorophyll a_I and its donor as well as an equilibration between different chlorophyll a_I molecules.

3. 3. Cytochrome f is also reduced by plastohydroquinone. A ratio of active cytochrome f to chlorophyll a_I of 0.4:1 is observed. The half-life time of the reduction of cytochrome f is 17 ms. The time course indicates that in the dark cytochrome f does not transfer electrons to chlorophyll a_I and that no more than 15% of the electron transport passes cytochrome f. Therefore cytochrome f should be situated in a side path of the linear electron transport.

4. 4. The electrons which are released from plastohydroquinone and are not accepted by oxidized cytochrome f and chlorophyll a_I have been calculated. From this difference properties of an electron carrier, as yet not identified, between plastoquinone and chlorophyll a_I are predicted.

The effect of T-2 toxin on bone microstructure in rabbits

T-2 toxin is the most toxic of the trichothecene mycotoxins. Its effect on bone microstructure is still unknown. This study focuses on acute effects of the T-2 toxin on compact and trabecular bone tissues structure of rabbits after a single intramuscular administration. Experimental E group (n?=?4) consisted of animals which were intramuscularly injected with T-2 toxin at dose 0.08 mg.kg^?1 body weight 72 h before slaughter. Group C (n?=?4) without T-2 toxin application served as a control. An absence of primary vascular longitudinal bone tissue near endosteal surfaces, its deposition on periosteal surfaces and a lower density of secondary osteons in the middle part of the substantia compacta were observed in both females and males injected with T-2 toxin. On the contrary, morphometrical analysis of the compact bone showed no demonstrable alternations in the sizes of primary osteons’ vascular canals, Haversian canals or secondary osteons between rabbits from E and C groups. Also, no significant effects of the T-2 toxin on trabecular bone morphometry and cortical bone thickness were observed between rabbits of either sex. The single intramuscular application of T-2 toxin at the dose used in our study affects only qualitative histological characteristics of the compact bone in rabbits.     

Effects of di-n-butyl phthalate on growth and photosynthesis in algae and on isolated organelles from higher plants

Di- n -butyl phthalate (DBF) is widely used as a plasticizer and has been found in all types of ecosystems. It inhibits growth and photosynthesis of green algae ( Chlorella emersonii CCAP strain 211/8 h and Selenastrum capricornutum CCAP strain 278/4) at concentrations higher than 10-⁵ M . The IC₅₀ value for CO₂-dependent oxygen evolution in algae was 3 × 10^-4M. The CO₂-reduction in isolated protoplasts prepared from barley ( Hordeum vulgare L. cv. Simba) was also inhibited by phthalate. The IC₅₀ value was 2 × 10^-4 M . The electron transport in isolated thylakoids prepared from spinach was inhibited with an IC₅₀ value of 3 × 10^-4 M . The IC₅₀ value for uncoupled electron transport extrapolated to zero chlorophyll concentration was 2.5 × 10^-5 M . The effect of di-n-butyl phthalate was localized to reactions in photosystem II. Di-n-butyl phthalate could thus be a pollutant which affects growth and photosynthesis of plants. The reported IC₅₀ values may be underestimated since di- n -butyl phthalate can attach to surfaces. The results are discussed in relation to observed effects of di- n -butyl phthalate on other organisms.     

Fine-Structure Locations of α-Glucan Phosphorylase, As Shown by Lead Precipitation and Electron Microscopy

Sites of glucan phosphorylase activity in fine structures, as shown by the lead precipitation method (Hori, Stain Techn., 39: 275, 1964) were studied by electron microscopy. Rat livers were fixed 2 hr at 0 C in buffered 2.5% glutaraldehyde, frozen-sections cut and incubated in the medium containing glucose-1-phosphate, 2.7 mM; NaF, 20 mM; acetate buffer, pH 5.8, 80 mM; Pb(NO₃)₂, 4.2 mM; and sucrose, 0.44 M; refixed in buffered 1% OsO₄, dehydrated and embedded in Epon 812 as usual. The reaction product was found in close association with endoplasmic reticulum, but not in mitochondria, nuclear membrane and the cisternae of endoplasmic reticulum. The possibility of demonstrating by the present method the indirect hydrolysis of glucose-1-phosphate through the phosphoglucomutase-glucose-6-phosphatase system was ruled out by inhibiting glucose-6-phosphatase with fluoride and ethanol.     

Uptake and light activated esterification of P by isolated chloroplasts

1. 1. Esterification of ³²P₁ by illuminated chloroplasts prepared on a sucrose gradient was examined to establish the optimal incubation conditions.

2. 2. The evidence is consistent with phosphorylation being closely coupled to the sum of noncyclic and pseudocyclic electron flow and with the rate of electron flow responding to the availability of electron acceptors.

3. 3. Apparent K_m values for ADP and Mg²⁺ were found to be 40 and 250 μM, respectively. The K_m value for Mg²⁺ was increased by the presence of Ca²⁺. Two apparent values were observed for P₁ at 0.2 and 1.1 mM. Chloroplast damage resulted in increased apparent K_m (P₁) values.

4. 4. Acceleration of the esterification resulting from the addition of ADP and P₁ to the medium indicated that these compounds were able to penetrate to the active site of esterification.

5. 5. Ribose 5-phosphate (Rib-5-P) was shown to inhibit P₁ esterification without affecting the apparent K_m for ADP or P₁. The evidence suggests that Rib-5-P interferes with the uptake of P₁, and possibly ADP.