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In vitro degradation of bone particles by human monocytes is decreased with the depletion of the vitamin K-dependent bone protein from the matrix.

The bone vitamin K-dependent protein osteocalcin has been suggested to play a role in bone resorption. By administering sodium warfarin to rats, it is possible to inhibit the vitamin K-dependent addition of the their gamma-carboxyglutamic acid residues to osteocalcin. This results in reduced amounts of osteocalcin bone, probably because devoid of the calcium-binding Gla residues, the protein no longer accumulates in bone. Preparations of bone obtained from rats treated with sodium warfarin for 6 weeks contained only 0.2% of normal levels of osteocalcin and were 90% reduced in the concentration of Gla. This bone could not be degraded by human monocytes in vitro as well as control bone (only 54% of control; P less than 0.003). Defects in the movement of cells to the bone were documented by phase contrast microscopy. Only 60% as many monocytes attached to the osteocalcin-depleted bone as to control bone in an in vitro attachment assay. These effects do not appear to be related to direct cellular toxicity. The degradation of bone in this in vitro system appears to be dependent on the osteocalcin content in matrix. This may result from defective movement of cells to bone and/or attachment to the bone.

Animals↗

Synthesis, characterization, and preliminary biological study of poly(3-(tert-butoxycarbonyl)-N-vinyl-2-pyrrolidone).

Poly(3-(tert-butoxycarbonyl)-N-vinyl-2-pyrrolidone) has been synthesized and characterized by gel permeation chromatography, Fourier transform infrared spectroscopy, NMR spectroscopy, and thermal analysis. The polymer is a chemically amplified photoresist. Arrays of lines with 25 microm width and 25 microm spacing were successfully patterned with this polymer by photolithography. Rat fibroblast cells were seeded on these patterned surfaces as well as the smooth glass surface. Phase contrast microscopy showed that cells on the patterned surfaces were strongly aligned and elongated along the grooves as compared to randomly spreading on the smooth surface. Since controlling cell orientation is critical for the development of advanced forms of tissue repair and cell engineering therapies, for example, peripheral nerve repair, production of tendon and ligament substitutes in vitro, and control of microvascular repair, the described polymer may be useful for applications in tissue reconstruction.

Animals↗

Hyposplenism in systemic lupus erythematosus.

Splenic function as measured by number of vacuolated red blood cells (RBC) seen with phase contrast microscopy was studied in 44 patients with systemic lupus erythematosus (SLE). Two of 44 patients (4.6%) had increased percentages of vacuolated RBC (8.4% and 30.6%) indicative of hyposplenism. Both patients had inactive disease at the time, were on low doses of corticosteroids, and had persistence of vacuolated RBC when restudied 2 and 4 months later. The present study confirms the association of hyposplenism with SLE and indicates that the incidence of hyposplenism in SLE is about 5%.

Adolescent↗

Measuring platelet function with platelet shape change, an early event in aggregation.

Platelet shape change precedes aggregation and is sensitive to significantly less stimulant. In this study, differences in percent light transmission (%T) through stirred suspensions of spheroid or discoid platelets are exploited for studying the response of platelets to Platelet Activating Factor (PAF). Changes in %T between stirred and non-stirred suspensions of unstimulated platelets in Platelet Rich Plasma (PRP) varied with the starting concentration. Aggregation was blocked by the presence of citrate while activation by ADP was minimized by apyrase. However, phosphatidic acid formation, ATP release and minimal serotonin release occurred. A shape change parameter (SCP) was defined which varied with the concentration of PAF. Its dependence on PAF was shifted to lower concentration with washed platelets. The dose-response curves for the SCP were found to be virtually identical for both sheep and human platelets. Human platelets were then used to correlate morphology, as determined by phase contrast microscopy, with spectrophotometric data in the form of the SCP. A linear regression analysis of SCP and percent activation revealed a virtual 1:1 correspondence with a slope of 1.066 and an intercept of 0.009 with the standard error of the estimate equal to 0.053. The correlation coefficient was 0.990, making it significant at p less than 0.001. This method provides an alternative to platelet aggregation assays as a convenient, sensitive and reliable measure of platelet activation.

Adenosine Triphosphate↗

Vascular histology of the guinea pig cochlea.

Detailed histological findings on all the regularly occurring cochlear vessels in the normal guinea pig are presented. Vessels were examined using a surface prepration technique and phase-contrast microscopy. Particular attention was paid to the density of red blood corpuscles, the thickness of the vascular wall, the occurrence of endothelial cells and pericytes, together with their possible influence on the vessel lumen and the existence of perivascular spaces and vessels lacking blood corpuscles. The data are compared with some previous findings of "vascular pathology" following noise stimulation. The possible functional significance of some of the morphological findings is also discussed.

Acoustic Stimulation↗

Hematuria in thin basement membrane nephropathy.

Thin basement membrane nephropathy (TBMN) often is diagnosed clinically when there is persistent dysmorphic or glomerular hematuria, but minimal proteinuria, normal kidney function, and no other obvious cause. This study investigated hematuria in patients with TBMN. A total of 112 patients with biopsy examination-proven TBMN were studied. All had hematuria at the time of presentation, with a mean urinary red blood cell (RBC) count of 256 +/- 250 x 10 3 /mL. Seventy-five (67%) patients attended for review over a median of 48 months (range, 3-120 mo) and provided a total of 485 urine specimens. Twenty-one patients (28%) had no hematuria by phase-contrast microscopy on at least 1 occasion. These corresponded to 32 urine specimens (7% of total). Of the 21 patients, the most recent urinary RBC counts were within the normal range in 11 (52%), but hematuria had recurred in the other 10 (48%). Hematuria is persistent in most patients with TBMN, but occasionally it resolves or is intermittent.

Diagnosis, Differential↗

Localization of endoplasmic reticulum in living and glutaraldehyde-fixed cells with fluorescent dyes.

Certain fluorescent dyes, previously reported to localize mitochondria, when used at higher concentrations also localize a continuous net-like structure in both living and glutaraldehyde-fixed cells. A similar reticular structure can be detected by phase-contrast microscopy and whole-mount electron microscopy in potassium permanganate-fixed cells as well. This structure is mostly tubular, with some patch-like areas, and is likely to be the endoplasmic reticulum (ER). The organization of the reticular structure is sensitive to colchicine and rotenone but not to cytochalasin B, taxol, monensin, the calcium ionophore A23187, 12-O-tetradecanoylphorbol 13-acetate, or hydrocortisone.

Animals↗

Anterior capsule opacification: a cell culture model.

We cultured the human lens epithelial cells with attaching anterior lens capsule using surgically removed capsular flaps obtained in intraocular lens implantation. The capsular flap was placed with cell side down in a small culture well. In phase contrast microscopy, an anti-meniscus plate was placed on the bathing medium to avoid optical aberration. The human lens epithelial cells showed outgrowth which ceased after 3 to 4 weeks of incubation. The cells under the capsule lost distinctive cell margin. The cells outgrew from the capsular edge both onto the anterior capsular surface and the well bottom. The outgrowth length and Bromodeoxyuridine uptake indicated a low proliferative potency of the human lens epithelial cells.

Cataract↗

[The preparation of fructose-modified chitosan microcarrier and culture of primary rat hepatocyte].

Crosslinked chitosan microcarries were prepared by the reaction of glutaraldehyde with fructose-modified chitosan. Various factors that influence the preparation were studied and the reaction conditions were optimized. Morphology of rat hepatocyte cultured on chitosan microcarriers was observed using phase contrast microscopy and scanning electron microscope, the metabolic activity was measured. Rat hepatocytes cultured on chitosan microcarrier retained the spherical shape as they have in vivo and had high metabolic activity. Fructose can enhance the metabolic activity of hepatocytes and fructose-modified chitosan microcarrier is a promising scaffold for hepatocytes attachment, which can be used in bioartificial liver support system.

Animals↗

[Prediction of spontaneous abortion by vaginal cytologic smears (author's transl)i1].

Vaginal smears were obtained from 175 women during the first four months of pregnancy weekly and the Karyopycnotic Index and the Eosinophilic Index encountered by phase contrast microscopy. The normal values were listed our by percentils. The development of KPI and Eo I during early pregnancy were compaired in three groups of patients: 1, 120 women with normal early pregnancy, 2.20 women with threatened abortion but good following up of later pregnancy, and 3. 35 women with spontaneous abortion. Even 4 weeks before abortion vaginal smears changed to pathological values in about 40% of all patients with spontaneous abortion. Women with threatened abortion but good outcome of pregnancy used to have normal KPI and Eo I.

Abortion, Threatened↗

[Morphology and deformability of erythrocytes in muscular dystrophy].

In recent years, the presence of red cell morphological abnormalities in patients with Muscular Dystrophy has made the object of numerous, often contradictory reports. A possible source of such confusion may lie in the fact that human erythrocytes are extremely sensitive to morphologic transformations resulting from various manipulations or environmental conditions in vitro. We have examined the morphology and deformability of erythrocytes from 7 patients with Duchenne and 9 patients with Steinert (myotonic) Muscular Dystrophy. To avoid preparation artifacts, fresh, unwashed red cells suspended in their own plasma were examined under phase contrast microscopy for the presence of either echinocytes and stomatocytes. Deformability was measured by filtration of dilute cell suspensions at constant flow rate through nucleopore membranes (nominal pore diameter = 3 micrometer). No significant difference was found between the patients' cells and those of 22 healthy volunteer controls. We conclude that previously reported abnormalities may have been the result of preparation artifacts. It appears possible, however, that erythrocytes from Muscular Dystrophy patients may be more sensitive than normal ones to certain stimuli originating from red cell manipulations in vitro.

Erythrocytes↗

Effects of some mechanical factors on the endospores of Bacillus subtilis.

Knaysi, Georges (Cornell University, Ithaca, N. Y.) and Harold R. Curran. Effects of some mechanical factors on the endospores of Bacillus subtilis. J. Bacteriol. 82:691-694. 1961.-Phase contrast microscopy was used to study incipient spore germination and early growth under conditions involving agitation with a platinum wire loop, sonic oscillation, and freezing and thawing. Treatment resulted in change in optical properties and destruction of a certain proportion of the spores. Up to one-tenth of the spores was destroyed by loop agitation. Sonic oscillation had little apparent effect upon incipient germination (darkening) of the spores, but subsequent development involving growth was stimulated in a considerable proportion. The effect of freezing and thawing upon incipient germination is discussed.

Bacillus subtilis↗

Effect of microtubule inhibitors on the expansion of hypoblast and margin of overgrowth of chick blastoderms.

The effect of the microtubule inhibitors, Nocodazole and taxol, was studied on the expansion of fragments of chick hypoblast (8 to 10 h incubation) and of margin of overgrowth (24 h incubation) cultured on artificial substrata and on the epiboly in intact blastoderms (24 h incubation). Immunocytochemical staining of these cells with antiserum against tubulin showed that 1 micrograms Nocodazole/ml caused disassembly of microtubules, and that 1 microgram taxol/ml led to increased but unordered assembly. The solvent dimethylsulphoxide had no effect. At these concentrations both microtubule inhibitors led to rapid arrest of the expansion of fragments of hypoblast and of margin of overgrowth in culture, and of the epiboly in intact blastoderms. Time-lapse films showed that inhibition of expansion in both situations was reversible within 2 h after removal of the drugs. Phase-contrast microscopy showed remarkably little difference between the morphology of treated as compared to untreated cultures. Measurements of the height of the cells on sectioned fragments of margin of overgrowth showed no differences between treated and untreated cultures. These results suggest that the cytoplasmic microtubule complex is important both for epiboly and for the migration of hypoblast cells in the chick blastoderm. The mechanisms of this microtubule-related migration are not understood.

Alkaloids↗

Chinese herbal medicines and their efficacy in treating renal stones.

In herbal treatment of kidney stones, antilithics are used to "dissolve" the stones or aid their passing to guard against further retention. Diuretic action is also needed to increase the amount of fluid going through the kidneys and flush out the deposits. Previous clinical studies have shown that herbal medicines and their concoctions could be used to inhibit calcium oxalate crystallization. However, the pharmacodynamics and in-vitro effects of such medicines have not been established. Five Chinese herbal medicines were selected based on their usefulness in treating stone disease. A 96-well plate oxalate-induced turbidity in artificial urine was used to evaluate the efficacies of the different herbal medicines on calcium oxalate crystallization. The metastable limit was determined and the nucleation rate was derived from 12-min time-course measurement of turbidity at 405 nm. Phase-contrast microscopy was used to visualize the crystals. The results showed that with increasing concentrations of herbal extracts, smaller calcium oxalate crystal sizes were observed. Overall, the five herbal medicinal extracts tested were able to promote nucleation of calcium oxalate crystals while at the same time decreasing the size. This in-vitro crystallization confirms that prophylaxis of renal stones could be achieved by reducing overall supersaturation through promotion of small crystal nucleates and concomitant pharmacological diuretic action of herbal medicines. Clinical studies will provide more definitive conclusions.

Calcium Oxalate↗

Polarized relationship of bacterial spore loci to the "old" and "new" ends of sporangia.

The frequency of association of spore loci with the "old" and "new" ends of rod-shaped sporangia in batch cultures of Bacillus megaterium ATCC 19213 was estimated by phase contrast microscopy. The analysis was facilitated by (i) the association of most of the sporangia into chains of two to five sporangia and (ii) the occurrence of two types of cross wall distinguishable by their degree of splitting. It was concluded that a newly formed spore is located at the "old" end of a sporangium. By inference, the sporulation division septum locus is distal to the ultimate normal cell division septum, i.e., proximal to the "old" pole of the B. megaterium sporangium. This result is discussed in relation to deoxyribonucleic acid segregation during sporulation.

Bacillus megaterium↗

Aspects of classification of Hemiptera hemocytes from six triatomine species.

The objective of this work was to characterize, and compare different morphological types of hemocytes of Rhodnius prolixus, Rhodnius robustus, Rhodnius neglectus, Triatoma infestans, Panstrongylus megistus, and Dipetalogaster maximus. This information provides the basis for studying the cellular immune systems of these insects. Seven morphological hemocyte types were identified by phase-contrast microscopy: prohemocytes, plasmatocytes, granular cells, cystocytes, oenocytoids, adipohemocytes and giant cells. All seven types of hemocytes are not present in every species. For example, adipohemocytes and oenocytoids were not observed in P. megistus and P. infestans, and giant cells were rarely found in any of the species studied. The hemocytes of Rhodnius and Dipetalogaster are more similar to each other than those from Triatoma and Panstronglus which in turn closely resemble each other. Emphasis is placed on methodological problems arising in this work which are discussed in detail.

Animals↗

Modulation of the growth and guidance of rat brain stem neurons using patterned extracellular matrix proteins.

Dissociated neuronal cultures on substrates patterned with extracellular matrix (ECM) proteins have yielded much information regarding the physiological characteristics of neuronal cells behaviour in vitro. However, neuronal patterning using long term embryonic brain slice cultures has not been comprehensively demonstrated to-date. Structuring was performed by micro contact printing of laminin. The slice cultures were evaluated by means of phase contrast microscopy at 3-22 days in culture. We were able to consistently achieve outgrowth of neurons, neurites and filopodia from brain stem slices cultured on ECM proteins structures of grid- and line-shapes. We believe that brain slice cultures on patterned substrates is a favourable approach to study functional synapses in vitro under defined conditions. The use of appropriate structures and the subsequent cell patterning may help to gain further understanding of axonal, dendritic and synaptic signal transductions and processes.

Animals↗

[Influence of substrates coated on cultured dorsal root ganglion cells].

Dorsal root ganglia from rat embryos were digested with trypsin to form single cell suspension. The cells were seeded on culture plates coated respectingly with poly-L-lysin (PLL), laminin (LN), PLL combined with LN, or collagen type I (CoI), then cultured in NB1 media. The cell survival and neurite outgrowth of cultured dorsal root ganglion cells were observed by phase-contrast microscopy. The results showed that the dorsal root ganglion neurons grown on PLL combined with LN were dispersively distributed at high survival rate, and those cells grown on CoI were clustered with thicker and longer neurites. It is suggested that the cell growth pattern could be influenced by different substances coated on the plates, and PLL combined with LN may provide a better substrum to culture and study single neuron soma and neurite.

Animals↗