Distribution of neurons in the major pelvic ganglion of the rat which supply the bladder,colon or penis

Summary In male rats a large number of the postganglionic neurons which innervate the pelvic organs are located in the major pelvic ganglion. In the present study we have identified the location within this ganglion of neurons which project to either of three pelvic organs, the penis, colon or urinary bladder. Two fluorescent retrogradely-transported dyes, Fast Blue and Fluoro-Gold, were used. For most animals one dye was injected into the cavernous space of the penis, the wall of the distal colon or the wall of the urinary bladder. In a small number of animals two organs were injected, each with a different dye. One to six weeks after injection the major pelvic ganglia were fixed in buffered formaldehyde. The distribution of fluorescent dye-labelled cells was observed in whole mounts of complete ganglia and, in most cases, also in small accessory ganglia located between the ureter and the prostate. The studies showed a unique pattern of distribution for each organ-specific group of neurons. Most of the colon neurons are located in the major pelvic ganglion near the entrance of the pelvic nerve, whereas almost all of the penis neurons are near or within the penile nerve. Bladder neurons are relatively evenly distributed throughout the ganglion. These results demonstrate a distinct topographical organization of organ-specific neurons of the major pelvic ganglion of the male rat, a phenomenon which has also been observed in other peripheral ganglia.     

Presence of serum albumin in normal human epidermis: Possible implications for the nutrition and physiology of stratified epithelia

In this paper, using both immunofluorescence and protein biochemistry techniques, we present definitive evidence that plasma proteins such as albumin are present within normal human epidermis. This result confirms several previous reports supporting the idea that relatively large molecules can diffuse through the epidermal basement membrane into epidermis. Our results bring new insights for discussing how hydrophobic ligands or drugs present in the bloodstream and bound to plasmatic carriers can reach epidermal cells of all layers.Abbreviations CHAPS 3-[3-cholamidopropyl dimethylammonio] propane sulfonate - kD kilodaltons - BSA bovine serum albumin - 2ME 2-mercaptoethanol - DTT dithiothreitol - SDS sodium dodecyl sulfate - pI isoelectric point - Mw molecular weight - Tris Tris-(hydroxymethyl) aminomethane - 1D one dimensional - 2D two dimensional - PAGE poly acrylamide gel electrophoresis - MEM Minimal Eagle's Medium     

Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Summary We have developed a serum-free medium for clonal growth of normal human muscle satellite cells (HMSC). It consists of an optimized nutrient medium MCDB 120, plus a serum-free supplement, designated SF, that contains epidermal growth factor (EGF), insulin, dexamethasone, bovine serum albumin, and fetuin. Fibroblast growth factor was needed with dialyzed fetal bovine serum (dFBS) as the only other supplement, but in media containing SF, it was only slightly beneficial, and was omitted from the final medium without significant loss. Clonal growth of HMSC in MCDB 120 plus SF is as good as with 15% serum and 0.5% chicken embryo or bovine pituitary extract. However, growth is further improved by use of a doubly-supplemented (DS) medium containing both SF and 5% dFBS. Clonal growth of HMSC in the DS medium far exceeds that in previous media with any amount of serum, and monolayer growth is at least equal to that in conventional media with higher levels of serum. Cells grown in these media exhibit little differentiation, even when grown to high densities. However, they retain the capacity for extensive fusion and synthesis of increased creatine kinase when transferred to a serum-free differentiation-promoting medium, such as Dulbecco's modified Eagle's medium plus insulin. All experiments were done with clonal cultures of HMSC to insure that observed growth responses were always those of muscle cells. This research was supported by a grant from the Muscular, Dystrophy Association. Editor's statement This article describes the optimization of both the basal nutrient medium and growth factor requirements for human muscle cells in vitro. This system is critical for studies of normal muscle cell and molecular biology, as well as for understanding diseases of muscle such as Duchenne, Muscular Dystrophy.     

Dependence of reaction rate of 5,5′-dithiobis-(2-nitrobenzoic acid) to free sulfhydryl groups of bovine serum albumin and ovalbumin on the protein conformations

The effect of protein conformations on the reaction rate of Ellman's reagent, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) with sulfhydryl (SH) groups of proteins was examined. The stopped-flow method was applied to follow the reaction of DTNB with SH group of two proteins, bovine serum albumin (BSA) and ovalbumin (OVA), at various concentrations of guanidine hydrochloride and urea. The rates for both the proteins were faster in guanidine than in urea. The rate sharply depended on the protein conformations, which were monitored by changes of helix contents on the basis of the circular dichroism measurements. The reaction rate of DTNB with SH groups of BSA was maximal around 2 M guanidine and 5 M urea. On the other hand, the reaction rate of DTNB with OVA was maximal at 3.5 M guanidine, while it gradually increased with an increase in the urea concentration. The amount of reactive SH group participating in the reaction with DTNB was also estimated by the absorbance change at 412 nm. The magnitudes of absorbance change for the reaction with free SH groups of OVA at low concentrations of the denaturants were appreciably smaller than those for BSA with one free SH group. Most of the four SH groups of OVA might react with DTNB above 5 M guanidine, although only a part of them did even at 9 M urea.     

Neutralizing effect of zinc oxide on dehydroabietic acid-induced toxicity on human polymorphonuclear leukocytes

The cytotoxic effect of dehydroabietic acid (DHAA), a resin acid found in rosin, was studied on human polymorphonuclear leukocytes using leakage of 51Cr from prelabeled cells, supravital staining, and transmission electron microscopy. DHAA caused a strong dose-related release of 51Cr, a high uptake of trypan blue, and total cell necrosis, as seen in transmission electron microscopy. Albumin slightly reduced the toxic effects, whereas the addition of zinc in various forms strongly inhibited these toxic effects of DHAA in the concentration range of 10-500 micrograms/mL. In the presence of albumin, zinc oxide as a suspension inhibited the damage of the cell membranes more than a filtrate of zinc oxide, indicating a subsequent slow release of zinc from the zinc oxide.     

Rotational correlation times of peptides determined by perturbed angular correlations of γ-rays

The technique of Perturbed Angular Correlations of -rays has been used to study the rotational correlation times in aqueous solution of the peptides: oxytocin, glycyltryptophan, cholecystokinin and the glycopeptide ristocetin. These peptides were labelled with excited ^111mCd through the covalent coupling of the metal chelator diethylenetriaminepentaacetic acid (DTPA) to the primary amines-of the peptides. The experimental correlation times are in good accordance with calculations based on the molecular weight. This indicates that the ^111mCd-DTPA is rigidly bound to the molecules. In the case of ristocetin, the correlation time was measured at 2°C, 25°C and 38°C. These experiments show the expected linear dependence on the viscosity divided by temperature. The feasibility of determining rotational correlation times for peptides without lysines and with correlation times in the ns region is thus demonstrated. Also, the correlation time of ^111mCd-DTPA coupled to the lysines of bovine serum albumin was determined. The measured correlation time is about 5 times less than the calculated correlation time. This effect is assigned to local motion. In spite of this, experiments show that ^111mCd-DTPA-bovine-serum-albumin is significantly immobilised by aggregation with immunoglobulins. The nuclear quadrupole interactions, necessary for determining the correlations times, were determined for ^111mCd-DTPA-ristocetin and ^111mCd-DTPA-bovine-serum-albumin by adding sucrose to a concentration of 63% and cooling to 2°C. This showed a small but significant difference between the two molecules. We interpret this as due to different conformations, possibly different coordination numbers. Offprint requests to: E. Danielsen     

Changes of fluorescence lifetime and rotational correlation time of bovine serum albumin labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride in guanidine and thermal denaturations

The nanosecond fluorescence depolarization method was applied to measure the fluorescence lifetime () and the rotational correlation time () of bovine serum albumin (BSA) labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl-Cl). Changes of and of dansyl BSA in the guanidine denaturation and in the thermal denaturation were examined. In parallel, the secondary structural change of dansyl BSA was followed by circular dichroism measurements. The magnitude of was almost unchanged between 1 and 2 M guanidine, where the secondary structure of the protein was predominantly disrupted; whereas that of began to increase before the disruption of secondary structure in the guanidine denaturation. In the thermal denaturation, in contrast, changes of both and occurred in a temperature range where the secondary structure was predominantly disrupted. The volume of equivalent sphere (V _e ) and the axial ratio () for the BSA were 3.6–3.8×10^–19 cm³ and 3.6 at 2M guanidine as against 2.1×10^–19 cm³ and 2.2 in the absence of guanidine (25°C), respectively. The magnitudes ofV _e and were 4.9×10^–19 cm³ and 4.5 at 65°C, respectively. Although the secondary structural change of dansyl BSA was irreversible in the thermal denaturation,V _e and were reversible.     

The major albumin protein from pea (Pisum sativum L.)

The major albumin protein in storage parenchyma tissue of developing peas has been localised at an ultrastructural level by immunocytochemistry. Tissue was fixed in buffered aldehyde and embedded in LR White resin which was polymerised by addition of catalyst. Sections were labelled by the indirect method of absorption of Protein A-gold to specifically bound antibodies. This method gives high levels of specific labelling on sections which retain good ultrastructural preservation and have high contrast after conventional staining. The albumin is located throughout the cytoplasm although no labelling was found associated with the endoplasmic reticulum, Golgi apparatus, vacuoles-protein bodies or other organelles.Abbreviation PMA pea major albumin protein     

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

Summary Human umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.