Improved microscopic detection of bacteriuria.

A simple method is described which permits the microscopic detection of bacteria in sediments of urine and other fluids, including bacteria that have eluded detection by conventional means. The method introduces increased centrifugal force and stepwise chemical fixation and then conventional staining. It is rapid, economical, and suitable for use in a physician's office. Use of this method immediately reveals those bacteria reported as "significant" by the conventional laboratory culture. More importantly, living or dead, which are missed by the conventional culture and by the conventional Gram staining procedure. These bacteria usually can be grown in special media and they appear to be related to systemic disease as evidenced by the clinical response to appropriate antibiotics.


Permanent stained preparations of synovial fluid for detection of calcium compounds using alizarin red S.

Permanent preparations of air dried synovial fluids were prepared by staining calcium compounds with alizarin red S stain; each slide was coverslipped with Permount. Variables studied were: (a) concentration of the solution of alizarin red S, (b) pH of staining solution, (c) time of incubation in staining solution and aqueous and ethanolic content of staining solution. The staining effect of each solution was tested on calcium pyrophosphate dihydrate, calcium oxalate, apatite and monosodium urate (MSU). Of all the solutions, best results were obtained with 0.25% alizarin red S in 50% ethanol at pH 7.0 for 30 min. With this solution, the calcium-containing compounds were well stained. MSU did not stain and still preserved negative birefringence on polarization. Fixation of smears with ethanol served a double purpose: It fixed the slides without dissolving or removing MSU or the calcium compounds, yet it did dissolve five corticosteroids commonly used for intra-articular injection which may interfere with interpretation of compensated polarized light microscopy of synovial fluids.


Basic blue 54: a new colorant for monocytes.

C.I. basic blue 54, a sulfur containing azo textile dye, stained the nucleus and cytoplasm of normal and leukemic monocytes bright red-violet. Essential for the staining reaction was a brief final rinse in a pH 3.6 acetic acid-sodium acetate buffer. Coloration of the type found in monocytes was not observed in other types of mature and immature leukocytes.


A simple staining procedure for detecting the true acrosome reaction in buffalo (Bubalus bubalis) spermatozoa.

A simple dual staining procedure for detecting the true acrosome reaction in dried smears of buffalo spermatozoa is described. Trypan blue is used first to differentiate live from dead spermatozoa and the dried smears which have been prepared are stained with Giemsa for acrosome evaluation. Four categories of spermatozoa were recognized: A) live, intact acrosome (acrosome pink, postnuclear cap clear); B) dead, intact acrosome (acrosome pink, postnuclear cap blue); C) live, detached acrosome (acrosome clear, postnuclear cap clear); and D) dead, detached acrosome (acrosome clear, postnuclear cap blue). The procedure is simple, rapid and convenient for assessing true acrosome reaction in buffalo spermatozoa. Simultaneous assessment of sperm viability and its acrosomal status in dried smears makes this procedure attractive because the true acrosome reaction can be studied thoroughly at a later state after the incubation period.


Microwave enhanced staining for plant virus inclusions.

Plant virus inclusion bodies can be stained specifically with established staining methods for light microscopy. The procedure can be augmented by a short microwave treatment to provide better staining intensity and reduced staining time. The method is useful for preliminary sampling prior to collection for electron microscopy and for plant pathologists, plant breeders, and diagnosticians as a rapid means of plant virus characterization.


Studies of the influence of different cross-linking reagents on the immune response against a B-epitope.

We have previously shown that the carrier polytuftsin obtained by polycondensation of tuftsin, a naturally occurring macrophage activator, increases significantly the antibody response against a linked B-epitope. In the present work, we have studied the influence of different cross-linking reagents on the quality of the conjugation and on the immune response, at both B-cell and T-cell levels. We observed that the cross-linking method used for coupling the B-epitope to the carrier influences the immune response. A hypothesis is put forward to explain the differences observed.


A technique for embedding undecalcified bone samples for detecting alpha-emitters using vacuum impregnation with Spurr's resin.

A method has been developed by which large samples of mineralized bone, containing an alpha-emitter, can be embedded in Spurr's resin in a fraction of the time required by conventional methods. Bone samples were freeze-dried or fixed and dried prior to impregnation with Spurr's resin under vacuum. Sections were cut for the preparation of either alpha-track or fission-track autoradiographs using the solid state detector CR-39. This method is applicable to samples containing a mobile form of a radionuclide that may be translocated during the processes of fixation and dehydration of the specimen.


New uses for calcium chloride solution as a mounting medium.

Fresh cross sections of stems (Psilotum nudum, Coleus blumei, and Pelargonium peltatum) and roots (Setcreasea purpurea) 120 microns thick were fixed in FPA50 (formalin: propionic acid: 50% ethanol, 5:5:90, v/v) for 24 hr and stored in 70% ethanol. The sections were transferred to water and then to 1% phloroglucin in 20% calcium chloride solution plus either hydrochloric, nitric, or lactic acid in the following ratios of phloroglucin-CaCl2 solution:acid: 25:4, 20:2, or 15:5. The sections were mounted on slides either in one of the three mixtures or in fresh 20% calcium chloride solution. A rapid reaction of the acid-phloroglucin with lignin produced a deep red color in tracheary elements and an orange-red color in sclerenchyma. Fixed and stored leaf pieces from Nymphaea odorata were autoclaved in lactic acid, washed in two changes of 95% ethanol, transferred to water, and treated with the three acid-phloroglucin-calcium chloride mixtures. The abundant astrosclereids stained an orange-red color similar to that of sclerenchyma in the sections. In addition, a new method is reported for specifically staining lignified tissues. When sections or leaf pieces are stained in aqueous 0.05% toluidine blue O, then placed in 20% calcium chloride solution, all tissues destain except those with lignified or partially lignified cell walls. Thus, toluidine blue O applied as described becomes a reliable specific test for lignin comparable to the acid-phloroglucin test.


Viability and acrosome staining of bull, boar and rabbit spermatozoa.

A practical and reliable staining procedure was developed to distinguish the viability and acrosomal status of bull, boar and rabbit spermatozoa. The first stain with trypan blue or Congo red is rapid and avoids artifacts. This stain is precipitated by neutral red during the 2 min required for fixation. The precipitate gives a high contrast black color, resistant to the subsequent rinsing and persists during the time required for staining the acrosome with Giemsa. Ten classes of spermatozoa are distinguished (live or dead with intact acrosomes, loose acrosomes, damaged acrosomes, no acrosome, or with no acrosome and no postacrosomal ring). The intact acrosomes are purple, the loose acrosomes are dark lavender and the damaged acrosomes are pale lavender. The anterior part of the head of live spermatozoa with no acrosome is white or light pink and the same area of dead spermatozoa is white or pale gray. The postacrosomal ring is red. The postacrosomal area of the head of live spermatozoa is white or light pink and the same part of dead spermatozoa is black, dark violet or gray. The procedure did not give satisfactory results for stallion spermatozoa.


Quantitative determination of murine dermal elastic fibers by color image analysis: comparison of three staining methods.

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Kligman's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.


Visualization of histamine binding to nuclei.

The binding of histamine to cultured microvascular endothelial cells and glycol methacrylate embedded ovarian tissue sections has been localized using fluorescein-albumin-histamine conjugate. Histamine conjugate was bound to the plasma membranes and nuclei of luteal, endothelial, and ovarian stromal cells. An apparent increase in the binding of histamine to nuclei was observed in the presence of cimetidine but the plasma membrane staining was still evident. Unlike cimetidine, pyrilamine completely inhibited the binding of histamine to the plasma membrane. Instead, in the presence of pyrilamine, histamine bound exclusively to the nuclei of endothelial, germinal epithelial, granulosa, and stromal cells. However, the nuclei of terminally differentiated luteal cells and oocytes were not labeled. The functional significance of these nuclear histamine binding sites remains to be determined.


Improved intracellular morphology of Pneumocystis carinii from rat lung by postfixation with a mixture of potassium ferrocyanide and osmium tetroxide.

Pneumocystis carinii infected rat lungs were postfixed with a mixture of OsO4 and K4Fe(CN)6. A marked improvement in staining of cell membranes, endoplasmic reticulum, nuclear membranes and glycogen was observed. These improvements were seen in both the trophic and cystic forms of the organisms. The addition of K4Fe(CN)6 did not improve the staining of cell walls, microtubules or ribosomes. Trophozoites were seen attached to both type 1 and type 2 pneumocytes by filopodia and/or intercalation of the cell body of P. carinii with the host lung cells. It is expected that the improvement in ultrastructural detail will allow better understanding of the ultrastructure of P. carinii and provide insights into the modes of action of various antimicrobial compounds on this organism.


A plastic embedding technique for analyzing fluorescent dextran-amine labelled neuronal profiles.

A plastic embedding technique employing fluorescently labelled dextran-amines is described. After application of tracer to cut nerves and appropriate transport time, animals were fixed in paraformaldehyde. Subsequently their brains were dissected, heads and brains were dehydrated, embedded in methacrylate and sectioned serially on a rotary microtome. Plastic sections allow high resolution of single neuron profiles and complete serial reconstruction of undistorted sections, including embryos with large amounts of yolk. In conjunction with whole mount analysis and double labelling, this technique can accurately reveal the spatial relationships of nerve components throughout development.


Identification of basic nuclear proteins by their boronate complex.

A new histochemical reaction for the identification of histone type basic proteins has been developed. Carbonyldiimidazol is used to activate the basic proteins of TCA-extracted nuclei, their m-aminophenylboronic acid complex is prepared, and the DNA-free, histone-containing nuclei are stained with toluidine blue at pH 5.5.


Embedding and fixation techniques for immunohistochemical staining with anti-DNA polymerase alpha and Ki-67 monoclonal antibodies to analyze the proliferative potential of tumors.

Anti-DNA polymerase alpha and Ki-67 are monoclonal antibodies that recognize nuclear antigens expressed in proliferating cells. In this study, we evaluated various methods of embedding and fixing brain tumor specimens to optimize staining with these antibodies. In fresh frozen sections, postfixation with 4% paraformaldehyde, 100% methanol, 95% ethanol and 10% buffered formalin were tested; also tested were prefixation with 4% paraformaldehyde followed by freezing and fixation with 100% methanol, 95% ethanol, or 10% buffered formalin followed by embedding in paraffin. For both antibodies, postfixation of fresh frozen sections with 4% paraformaldehyde at 4 C gave the most intense staining and lowest background activity while preserving histological features. This technique can be used in routine clinical practice to predict the growth potential of tumors.


Effects of prostanoids on the rat's myometrium in vitro during pregnancy.

The objectives of this study were to determine the effects of pregnancy in the rat on the contractile response of the myometrium in vitro to a number of prostanoids. Longitudinally and circularly oriented strips were studied separately. Responses to PG (prostaglandin)F2 alpha, PDG2, the PGI2-mimetic iloprost, and the thromboxane (Tx) A2-mimetic U-46619 were investigated on Days 10, 15, 18, 20, 21, and 22 of pregnancy. Responses were prostanoid-dependent, and differences between longitudinal and circular strips were small. PGF2 alpha and PGD2 produced similar patterns, with a high potency but low maximal response on Day 10; thereafter potency fell to a minimum value on Day 18 and then gradually increased until Day 22, when it was still lower than at Day 10. In contrast, for PGE2 there were no changes in potency over the period of study (longitudinal muscle) or a slight increase between Days 15 and 21 (circular muscle). Both iloprost and U-46619 maintained a low potency throughout pregnancy. We conclude that the pregnant rat's myometrium is probably not a target for PGI2 or TxA2 and that the difference patterns of responses to PGE2 and PGF2 alpha during pregnancy support the hypotheses that these prostanoids act at different sites within the myometrium.


Voltage-gated calcium channels in rat Sertoli cells.

We have studied Ca2+ voltage-gated channels of immature rat Sertoli cells by measuring intracellular Ca2+ concentration and its variation following administration of various agents in fura-2-loaded, confluent monolayers in culture. Our findings indicate that the basal Ca2+ intracellular level is about 100 nM, a value that falls within the range found in most eukaryotic cells. The intracellular Ca2+ level is rapidly increased by fetal bovine serum through release of intracellularly stored Ca2+ and opening of membrane cation channels. Substantial Ca2+ influx in rat Sertoli cells seems to be mediated by voltage-gated cation channels, which are sensitive to nifedipine, nicardipine, and omega-conotoxin. To investigate whether FSH, which controls several morphological and biochemical events of prepubertal Sertoli cells, modified Ca2+ influx in this cell type, we analyzed the cell response to acute FSH administration. The results show that, although not influencing the basal concentration of Ca2+, FSH decreases intracellular calcium influx induced by membrane depolarization. Similar data were also obtained by adding dibutyryl cAMP to the external medium and by increasing endogenous cAMP.


Steady-state amounts of alpha- and luteinizing hormone (LH) beta-subunit messenger ribonucleic acids are uncoupled from pulsatility of LH secretion during sexual maturation of the heifer.

Our primary objective for this study was to determine whether steady-state amounts of alpha- and LH beta-subunit mRNAs in the anterior pituitary are altered during sexual maturation in the bovine female. A secondary objective was to determine whether 17 beta-estradiol (E2) alters amounts of LH subunit mRNAs before onset of puberty. Heifers (7 mo old) were assigned to one of three treatments: 1) ovariectomized (OVX, n = 16); 2) OVX and administered E2 (OVXE, n = 16); or 3) ovary-intact (INTACT, n = 20). Pituitaries were collected at an estimated 120 days before onset of puberty (prepuberty) or 25 days before onset of puberty (peripuberty). Six INTACT heifers were used to determine time of puberty during the experimental period, and their pituitaries were collected 40 h after administration of prostaglandin F2 alpha (postpubertal INTACT group). Relative amounts of mRNAs for LH subunits in each pituitary were determined by Northern analysis and scanning densitometry. Amounts of alpha- and LH beta-subunit mRNAs were lower in pituitaries of INTACT heifers and OVXE heifers, regardless of stage of sexual maturation, than in those of OVX heifers. Amounts of alpha-subunit mRNA were similar in OVXE and INTACT heifers regardless of stage of sexual maturation. Amounts of LH beta-subunit mRNA did not change during sexual maturation in heifers in the INTACT group. Concentrations of E2 were higher and LH beta-subunit mRNA were lower in heifers from the prepubertal OVXE group than in heifers in all other treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)


Anti-RNA polymerase I antibodies in the sera of MRL lupus mice at the initial stages of disease are directed primarily against phosphorylation-dependent epitopes.

Anti-RNA polymerase I (RPI) antibodies in the sera of MRL/Mp-lpr/lpr and MRL/Mp(-)+/+ mice, which develop an autoimmune disease similar to human systemic lupus erythematosus, were screened for reactivity with purified RPI or RPI which had been dephosphorylated. In every case (n = 10), dephosphorylation of RPI resulted in a significant decrease (33-95%) in antibody binding. The anti-RPI antibodies in the sera of the same mice approximately 6 weeks later also reacted better with untreated as compared to dephosphorylated RPI but, in every case, the decrease in antibody (0-30%) caused by dephosphorylation was substantially diminished. That the proportion of anti-RPI antibodies in the sera of MRL mice decreased with progression of lupus-like disease was confirmed by closely monitoring the antibodies over the course of disease. Anti-RPI antibodies produced at the earliest stages appeared to be directed almost exclusively against phosphorylation-dependent determinants since dephosphorylation of RPI essentially abolished antibody binding. Subsequently, the percentage of the total anti-RPI antibodies in the sera of these mice directed towards phosphorylation-independent epitopes increased linearly with time. The importance of phosphorylation-dependent epitopes on RPI for the development of the anti-RPI autoimmune response was supported by the observation that treatment of mice with alkaline phosphatase partially attenuated anti-RPI antibody production.


Anti-insulin antibody structure and conformation. I. Molecular modeling and mechanics of an insulin antibody.

A knowledge-based three-dimensional model of an anti-insulin antibody, 125, was constructed using the structures of conserved residues found in other known crystallographic immunoglobulins. Molecular modeling and mechanics were done with the 125 amino acid sequences using QUANTA and CHARMm on a Silicon Graphics 4D70GT workstation. A minimal model was made by scaffolding using crystallography coordinates of the antibody HyHEL-5, because it had the highest amino acid sequence homology with 125 (84% light chain, 65% heavy chain). The three hypervariable loop turns that are longer in 125 than in HyHEL-5 (L1, L3, and H3) were modeled separately and incorporated into the HyHEL-5 structure; then other amino acid substitutions were made and torsions optimized. The 125 model maintains all the structural attributes of an antibody and the structures conserved in known antibodies. Although there are many polar amino acids (especially serines) in this site, the overall van der Waals surface shape is determined by positions of aromatic side chains. Based on this model, it is suggested that hydrogen bonding may be key in the interaction between the human insulin A chain loop antigenic epitope and 125.


The interactions of neuropeptides with membrane model systems: a case study.

The interactions between the positively charged neuropeptides substance P (SP), bradykinin (BK), and zwitterionic Met-enkephalin (ME) neuropeptides, and negatively charged SDS and zwitterionic lysophosphatidylcholine (LPC) membrane model systems, have been investigated using one- and two-dimensional nmr experiments. Proton longitudinal relaxation studies were used to characterize these interactions as intrinsic or extrinsic. An extrinsic interaction are similar to those observed for extrinsic membrane proteins. An intrinsic interaction are similar to those observed for intrinsic membrane proteins, and would require that the hydrophobic residues penetrate or insert into the hydrophobic core of the membrane. The interactions between both SP and BK and SDS, based on nmr results, may be characterized as intrinsic, and the interaction between ME and SDS may be characterized as extrinsic. Two-dimensional nuclear Overhauser enhancement spectroscopy experiments proved the insertion of the phenylalanine residues on both SP and BK into the hydrophobic core of SDS micelles. The interaction between SP and BK with LPC based on nmr results are characterized as extrinsic, with the interaction between ME and SDS characterized as weakly intrinsic.


Interleukin 4 amplifies monocyte chemotactic protein and interleukin 6 production by endothelial cells.

Upon exposure to interleukin 4 (IL-4), human umbilical vein endothelial cells (EC) produced low amounts of IL-6 and expressed low levels of IL-6 transcripts. While being a weak stimulus for IL-6 production, IL-4 amplified the release and mRNA expression of this cytokine in response to optimal concentrations of IL-1. IL-4 also induced low levels of monocyte chemotactic protein but amplified the expression of this chemoattractant in response to inflammatory signals. Thus IL-4, by amplifying at the level of the vascular endothelium the expression of vascular cell adhesion molecule 1 selectively, as well as that of chemoattractants and IL-6, may favour the selective recruitment and activation of lymphoid and monocytic elements crucial for subacute/chronic inflammation and immune reactions.


Inhibitory effect of recombinant intracellular interleukin 1 receptor antagonist on endothelial cell activation.

This investigation was designed to elucidate whether an intracellular version of interleukin 1 receptor antagonist (icIL-1ra) interferes with the action of IL-1 at the level of vascular cells. Recombinant icIL-1ra inhibited the IL-1-induced production of IL-6, IL-8 and monocyte chemotactic protein by human endothelial cells (HEC). Moreover, icIL-1ra inhibited induction of adhesion molecules by IL-1. Endotoxin lipopolysaccharide (LPS), an IL-1 inducer, stimulated a spectrum of functions in EC similar to that activated by IL-1, but icIL-1ra did not interfere with the LPS activation of EC. This observation suggests that induction of extracellular IL-1 is not an important intermediate event in the response of EC to LPS. Unlike LPS-stimulated monocytes, EC exposed to different inducers did not express appreciable levels of IL-1ra mRNA transcripts as assessed by northern blot analysis. IL-1ra produced by mononuclear phagocytes, represents a negative regulator circuit of the action of IL-1 on EC and could be important in the control of vascular participation in inflammation and immunity.


[Metallic mesh endoprosthesis and intraluminal high dose rate 192Ir brachytherapy in the palliative treatment of malignant bile duct obstruction. Initial results].

Since December 1989, 9 patients with inoperable malignant biliary tract obstruction were treated palliatively by a combined modality treatment consisting of placement of a permanent biliary endoprosthesis followed by intraluminal high dose-rate 192Ir brachytherapy. A dose of 10 Gy was delivered in a hyperfractionated schedule at the point of reference in a distance of 7.5 mm of centre of the source. External small field radiotherapy (50.4 Gy, 1.8 Gy per day, 5 fractions per week) was also given in six cases (M/O, Karnofsky greater than 60%). In 9/9 cases an unrestrained bile flow and an interruption of pruritus was achieved, in 78% (7/9) of cases the duration of palliation was as long as the survival time (median survival time 7.5 months).


Immune surveillance against Epstein-Barr virus.

Following primary infection, EBV retains a life-long latent association with B lymphocytes and a permissive association with stratified epithelium in the oropharynx. This review presents a model for the host-virus relationships in healthy virus carriers, a relationship which, if perturbed, may result in EBV-associated disease. Cytotoxic T cells that recognise virally-determined epitopes on infected cells are the major effector arm and control the persistant infection. A strategy for developing a vaccine to EBV is discussed.


Deployment of extracellular matrix proteins in sea urchin embryogenesis.

The apical extracellular matrix of the sea urchin embryo, known as the hyaline layer (HL), is a multi-laminate organelle composed of at least 10 polypeptides. Although integrated into one ECM, HL proteins exhibit individual temporal and spatial dynamics throughout development. These molecules are stockpiled in the oocyte during vitellogenesis in at least four distinct vesicle populations. They are released onto the cell surface at fertilization in a specific order, and interact differentially with embryonic cells as development proceeds. Many experiments have suggested that the HL is vital for embryogenesis, but relatively little is known about the functions and interactions of its constituent molecules. The purpose of the present review has been to gather information on the basic characteristics of the known HL proteins together with data on their expression in the embryo, and where possible, their biological activities. Compiled, these observations may provide some insight into the workings of a uniquely embryonic organelle.


Characterization of insulin-like growth factor receptors in the insulin-responsive R3230AC mammary adenocarcinoma.

To ascertain whether insulin-like growth factors (IGF-1 and IGF-2) affect the estrogen- and insulin-responsive R3230AC mammary carcinoma, studies of IGF-1 and IGF-2 receptors were conducted on primary-culture tumor cells and on membranes purified from whole tumors. By saturation binding analysis, cells in culture displayed one class of IGF-1 binding sites with an affinity constant, Kd, of 5.5 +/- 0.7 x 10(-9) M, whereas in membrane preparations, high and low affinity IGF-1 binding sites, with Kd's of 5 +/- 1.7 x 10(-9) M and 1 +/- 0.4 x 10(-7) M, respectively, were detected. Specificity of binding was demonstrated by 85% displacement of 125I-IGF-1 with 1,000-fold excess unlabeled IGF-1 and 50% displacement with 6.5 x 10(-9) M IGF-1 or 3 microM insulin. Binding sites for IGF-2 were also demonstrable in cultured cells, having a Kd of 7 +/- 0.8 x 10(-10) M, and 50% displacement was obtained with 1.5 x 10(-9) M IGF-2 or 1.5 x 10(-8) M IGF-1. Cross-linking experiments on cultured cells confirmed the presence of IGF-1 and IGF-2 receptors. In purified tumor membranes, IGF binding proteins (M(r) 28,000-32,000) were also detected; their labeling was not displaced by 10(-5) M insulin. In vitro, the tumor cells secrete one or more IGF binding proteins into the medium. Despite the fact that these cells expressed specific IGF receptors, their growth was apparently independent of these growth factors, since neither IGF-1 nor IGF-2 was mitogenic for R3230AC cells in vitro. Nevertheless, IGF-1 caused concentration-related significant increases in the plating efficiency of these cells. Further studies are necessary to determine the functional role of these growth factors, their receptors, and their binding proteins in the biology of this rodent mammary tumor.


Endothelial cell migration and chemotaxis in angiogenesis.

Our goal has been to provide a quantitative analysis of the random motility and chemotaxis of microvessel endothelial cells (MEC) in order to understand the role of these functions in the development of new capillaries. A major difference of our work from previous investigations has been our use of mathematical analysis to interpret experimental results, and to relate in vitro migration measurements to in vivo angiogenesis observations. In this paper we present some of our methods and their rationale, with recent results from both experiment and mathematical models.


Differential expression of integrin cell-substratum adhesion receptors on endothelium.

The interactions of the endothelial cells with their underlying extracellular matrix is of fundamental importance to angiogenesis. Therefore, the distribution of integrin cell-substratum adhesion receptors on cultured and in situ endothelial cells was determined. Cultured endothelium from large and small vessels and large vessel endothelial cells in situ expressed integrins that bound to both basement membrane and inflammatory matrix components. In contrast, in situ microvessels primarily expressed basement membrane binding integrins. Alterations in the expression of the integrins on microvessel endothelial cells may be important for microvascular remodeling during wound healing and/or angiogenesis.


Studies on basic fibroblast growth factor (FGF-beta) gene expression in the rat and pig ovary using in situ hybridization and quantitative reverse transcriptase--polymerase chain reaction techniques.

In order to gain further understanding of the physiology of basic fibroblast growth factor (FGF-beta) in the mammalian reproductive tract, the expression of FGF-beta mRNA in the rat and porcine ovary has been examined by in situ hybridization and quantitative reverse transcriptase-polymerase chain reaction techniques during different stages of the estrus cycle. The results confirm that an increase in FGF-beta mRNA levels occurs over the course of the estrus cycle. No FGF-beta gene expression was detected during diestrus, the non-hormonal phase of the cycle, or at the early proestrus stage of the cycle. During late proestrus and estrus, FGF-beta mRNA was predominantly localized to granulosa cells of the dominant follicles, and to a lesser extent, to secondary antral follicles not committed to ovulation. These cells also expressed FGF-beta mRNA during this phase of follicular development, albeit in low abundance. During metestrus, after ovulation, in the newly formed corpora lutea FGF-beta mRNA levels were maximal, however on entering the next cycle commencing at diestrus, no FGF-beta mRNA was observed in the degenerating corpora lutea. These results indicate that expression of the FGF-beta gene is differentially regulated during the estrus cycle. The biological significance of this expression and the potential role of FGF-beta in local intra-ovarian regulation of the repetitive cycles of follicular differentiation, proliferation and maturation associated with ovarian revascularization are discussed.


Angiogenesis--biomedical technology.

All of these studies show that angiogenesis research can benefit from new biomedical technology tools currently being developed, as well as contribute by providing new technologies that can be used in other areas of medicine. It is hoped that the chapters in this book in this area will provide the reader with an up-to-date appreciation of some of the exciting research that is currently being pursued.


Two cloned cerebral endothelial cell phenotypes: an in vitro model for angiogenesis?

Expression of smooth muscle alpha-actin and migratory behaviour of cloned cerebral endothelial cells (cEC) which exhibited two distinct phenotypes (type I, type II) were studied. Removal of mitogenic factors (alpha ECGF, ECGS) and heparin from the culture medium resulted in a smooth muscle-like appearance (type II) of the cells, expression of smooth muscle alpha-actin protein and smooth muscle actin mRNA and in an increased migratory activity. In contrast, addition of growth factors and heparin led to a cobblestone-like phenotype (type I) which lacked the expression of smooth muscle alpha-actin but expressed other proteins as determined by 2-D-gel electrophoresis.


Tumor cell spheroids induce a mitogenic response in endothelial cells.

Conditioned media from spheroids of human colorectal tumor cell lines HT29, SW620, SW480 and CaCo-2 were tested for their mitogenic activity on human umbilical vein endothelial (HUVE) and bovine capillary endothelial (BCE) cells. All spheroid-conditioned media were capable of increasing the labeling indices in both endothelial cell populations, with the mitogenic activity decreasing in the following order: SW620, HT29/SW480 and CaCo-2. The endothelial cell mitogenic activity of the spheroids was not dependent on the presence of necrosis nor on their in vivo cytokinetic characteristics. We conclude that tumor spheroids release mitogenic activity for endothelial cells, but this stimulation depends on the age of the tumor spheroid as well as on the type of tumor used.


On the quantitative rat mesenteric-window angiogenesis assay.

The rodent mesenteric-window angiogenesis assay permits the quantitative assessment of angiogenesis in adult, normally-vascularized mammalian tissue in the intact animal. Using appropriate optical magnifications, even very small, newly-formed vessels are recorded. The ultimate value of the model will, of course, depend on how pertinent it is to the features which govern angiogenesis in the tissues that are involved in clinically-relevant, angiogenesis-related diseases.


Wound healing in aged animals--effects of locally applied transforming growth factor beta 2 in different model systems.

Local application of a growth factor which could stimulate cell turnover, extracellular matrix synthesis and blood vessel formation in the skin should improve and accelerate wound healing processes which are often impaired in old age. We demonstrate the effects of TGF-beta 2 in promoting wound repair in old animals where normal healing responses are shown to be naturally slower. The potential use of TGF-beta s for the treatment of wound injuries, including chronic non-healing ulcers in the elderly, is discussed.


Delivery systems for angiogenesis stimulators and inhibitors.

The development of biocompatible, controlled release systems for macromolecules has provided the opportunity to deliver angiogenesis stimulators and inhibitors. These systems have also facilitated the purification and characterization of a number of these substances. In this paper, we discuss controlled release delivery systems which release angiogenesis factors through either porous polymer matrices, degradable polymeric delivery systems or modulated polymer release systems.


Angiopolarity of cell carriers: directional angiogenesis in resorbable liver cell transplantation devices.

The purpose of this study was to obtain directional angiogenesis of small blood vessels and capillaries to an implant made from a resorbable polymer for hepatocyte transplantation. It was intended to mimic the native acinar structure of the liver in order to facilitate replication of the cells and organ growth. The implant device structure was designed for injection to minimize surgical trauma. Hollow microspheres with an open porous wall structure and one large central opening were made from poly(d,l-lactic-co-glycolic acid) (85:15 lactic:glycolic). This polymeric scaffold was seeded with hepatocytes and implanted into the abdominal wall muscle of syngeneic Fisher rats. Specimens explanted up to 56 days p.o. showed hepatocyte survival and the development of a directional blood supply. This phenomenon is coined "angiopolarity". The study should help in addressing the issue as to whether avascular cell implants with post-transplantation organ growth should be attempted. Processing options in applying heat to the polymer solution allow manufacturing of larger microspheres with different diameters of central openings. This would allow the use of the scaffold for other cell transplantations than hepatocytes.


Clinical investigations on blood perfusion in human malignancies of the pelvis and abdomen: significance for tumor therapy.

Blood flow of deep pelvic and abdominal tumors was investigated with the thermal clearance method, dynamic CT and dynamic MRI. There are good correlations between the measurement values obtained by these methods. A low flow was observed in rectal cancer and soft tissue sarcoma in contrast to pancreatic cancer and hypernephroma. The temperature increase induced by regional hyperthermia was dependent on the individual tumor blood flow. Dynamic CT can be used pretherapeutically and predict the quality of a heat treatment, which is important with regard to concepts consisting in radiotherapy or chemotherapy plus hyperthermia.


Induction of angiogenesis by growth factors: relevance to pancreatic islet transplantation.

Biodegradable pellets releasing 20 ng/day of endothelial cell growth factor alpha (alpha ECGF) or a- or b-fibroblast growth factor (FGF) and 90 micrograms/day of heparin were implanted beneath the renal capsule in rats and dogs and the muscularis/serosal border of the pyloric stomach in dogs to test for angiogenesis in a potential pancreatic islet transplant site. These factors were also tested in vitro to determine whether the capillary bed of the isolated islet could be preserved. alpha ECGF was superior to a- or bFGF in promoting endothelial cell growth and capillary formation in isolated islets. Both a- or bFGF and alpha ECGF induced the development of a dense capillary bed in the dog stomach, whereas in the kidney site alpha ECGF was more effective in the rat than was a- or bFGF. Priming the isolated islet as well as the transplant site prior to islet transplantation resulted in islet blood flow being established within 3 days in contrast to 7-14 days in controls.


Capillary growth in the heart.

Experiments were performed to test the hypothesis that increased stretch and/or tension of myocytes in the absence of changes in blood flow could induce capillary growth in the heart. Chronic treatment with either dobutamine (rabbits) or alinidine (rats) which increased force of contraction and/or stroke volume respectively without increases in coronary blood flow led to enlargement of the anatomical size of the capillary bed, with no change in cardiac weight, thus supporting the role of external mechanical factors in angiogenesis.


Endothelial cell proliferation is induced by radiation in cultured explants of human urothelium and oesophageal mucosa.

Normal oesophageal mucosa obtained during upper abdominal surgery or urothelium obtained from kidney transplants was placed in explant culture and exposed to 60Co gamma radiation after 48 h. Cultures were maintained for two to six weeks after exposure and were monitored at various intervals for the development of features associated with malignant transformation. Endpoints examined included proliferation rate, frequency of proliferating cells, cell type distribution and degree of differentiation of the different cell types. The results indicate that following exposure to gamma rays (2.5-10 Gy) an increased overall growth rate of the surviving cells can be observed 2-4 weeks later. Analysis of the results using autoradiography confirms that a higher level of cell proliferation occurs in treated cultures than in the control untreated cultures. When the distribution of different cell types in the culture is examined, the increase in growth can be seen to be due to greatly increased numbers of endothelial cells. These proliferated over the surface of the epithelial cells and are more strongly positive for endothelial cells markers than endothelial cells occurring in control cultures. The degree of differentiation of endothelial cells into capillary like structures is also more apparent in carcinogen treated cultures. Foci expressing both epithelial and endothelial markers also occur. The results suggest that exposure of tissue fragments to radiation stimulates the growth and development of endothelial cells in resulting cell cultures. The effect may be due to a direct action of the treatment on the endothelial cells but it is more likely that it results from a secondary effect mediated by traumatic response of damaged epithelial cells.


Studies on tumor induced angiogenesis.

Methods were developed to test angiogenic response to human tumor implants and various biologic agents in the cornea of rabbits and non-human primates (Macaca arctoides). Crude PDGF preparations were found to have significant angiogenic effect. Purified, recombinant PDGF preparations were also effective inhibitors (e.g. pentoxifylline (Px) (which also were found to release PgI2 and t-PA) inhibited human tumor implant induced angiogenesis and reduced spontaneous metastases in 3 transplantable murine tumors (Furth-Columbia Wilms' tumor in Furth-Wistar rats, C-1300 neuroblastoma in A/J mice and HM-Kim mammary carcinoma in Wistar rats) but not in the NIH adenocarcinoma in Balb/c mice. Sodium diethyldithiocarbamate (DDTC), a metal complexing agent with special affinity to copper and anti-thyroid as well as, immune stimulating activity was shown to be anti-angiogenic and to potentiate the effect of Px. The anti-fibrinolytic agents epsilon amino caproic acid (EACA) and tranaxamic acid (t-AMCHA) were anti-angiogenic. DDTC and Px were synergistic from this point of view.


Angiostatic activity of anticancer agents in the chick embryo chorioallantoic membrane (CHE-CAM) assay.

Most anticancer agents are screened for antiproliferative and differentiation-inducing activity on tumor cells, but not for differential effects on vascular endothelium. Clinically active cytotoxic or cytostatic agents and selected analogs were tested in a closed, ethically accepted and inexpensive in vivo fertilized egg system [CHE-CAM]. The results revealed characteristic effects on vessel numbers between day 7 and 11 and on embryo weight at day 12. Hitherto unknown angiostatic activities were discovered with antitumor antibiotics, plant alkaloids, antimetabolites and other classes of compounds. The information provided by the CHE-CAM assay could represent a valuable complementation to the existing drug-screening systems for solid tumors and suggests a selective angiostatic role of some antitumor agents currently used in combination treatments.


In vitro activity of novel sulphonic derivatives of distamycin A.

The successful growth of tumors is dependent on the process of vascularization elicited by the tumor itself. As confirmed by many authors, there is a correlation between the presence of factors that stimulate tumor growth and angiogenesis. One of the approaches we have explored to control angiogenesis has been to synthesize compounds able to complex growth factors. A number of sulphonated derivatives of distamycin A were found active in inhibiting the binding of bFGF and PDGF beta on Swiss 3T3 cells with ID50 values ranging between 142-587 microM for bFGF and 28-79 microM for PDGF beta. The effect of these new derivatives in inhibiting angiogenesis was initially explored in the chorioallantoic membrane assay. It was observed that the selected compounds were active in this model system at the concentration of 350 nm/pellet. These new molecules present low or no cytotoxic activity on M5076 murine reticulosarcoma cells, the ID50 values being higher than 60 microM after 72 h continuous exposure in vitro.


In vivo activity of novel sulphonic derivatives of distamycin A.

Solid tumor growth can be modulated through inhibition of vascularization elicited by angiogenic factors. With the objective to complex these factors, new derivatives of distamycin A were synthesized and evaluated in vitro [1] and in vivo for their ability, after i.v. administration, to inhibit bFGF-induced vascularization and the growth of M5076 murine reticulosarcoma implanted i.m. The tested compounds were able to block angiogenesis with inhibition values ranging between 70-100%. Moreover, they were found to be capable of inducing tumor inhibition with values ranging between 40% and 95% at non-toxic doses.


Maturation of newly-formed subretinal vessels.

The maturation process of newly-formed subretinal vessels was studied ultrastructurally. Primitive new vessels have thick endothelial cells with cytoplasm that contains many ribosomes and rough endoplasmic reticulum. Some immature endothelial cells show budding, and many of the intercellular junctions have small gaps. As the newly-formed vessels mature, the endothelial cells become attenuated and fenestrated, and their junctions become tight. Mature subretinal new vessels closely resemble those of the normal choriocapillaris.


Embryonic development of the CNS microvasculature in the mouse: new insights into the structural mechanisms of early angiogenesis.

Vascularization and expression of blood-brain barrier (bbb) associated morphological characteristics were studied by ultrastructural analysis during the embryonic development of the mouse CNS. At day 9 (E9) capillaries were only found in the perineural mesenchymal tissue exhibiting fenestrations and loosely attached pericytes. At E10 endothelial cells (EC) together with pericytes and other mesenchymal cells invaded the intraneural domain. The immigrating EC showed numerous protrusions and lost their fenestrations as soon as the new intraneural capillaries were formed. Intraneural EC showed prominent nuclei, many pinocytotic vesicles and junctional complexes which appeared tight in some places. These results indicate that from the first day of intraneural vascularization onwards, the morphological properties of the bbb are present in the early embryonic mouse cerebral cortex.


Tumor angiogenesis: evidence of new blood channels from plasma infiltrations.

Stroma formation in Ehrlich carcinoma, studied with histochemical and TEM techniques, is similar to wound healing. In this tumour mast cells, macrophages, adipocytes, platelets and fibrin seem to co-operate locally with malignant cells in regulating stroma formation. The gaps opened in the tumor parenchyma by plasma outpouring from local blood vessels seem to offer easy routes for endothelial cell migration towards ill-nourished areas, and may explain the irregular aspect of tumor microvascularity.


Hemopoietic stem cell processing: comparison of progenitor cell recovery using the Cobe 2991 cell washer and the Haemonetics V50 apheresis system.

Using 24 bone marrow (BM) harvests intended for cryopreservation and transplantation, we compared the use of the Cobe 2991 cell washer (2991) and the Haemonetics V50 apheresis system (HV50) for automated BM processing. Our in vitro data indicate that while the mononuclear cell (MNC) concentration of the HV50 product was significantly greater than that of the 2991, the overall MNC recovery of the two products was equivalent. In addition, although the concentration of CFU-GM and BFU-E in the products was equivalent, recovery of these progenitors in the 2991 product was significantly greater than those of the HV50 product. There was no significant difference in either the final product concentration or the overall recovery of cells bearing the primitive myeloid antigens, CD33 or CD34, between the two methods. The HV50 product volume, the red cell and the granulocyte mass were significantly lower than those of the 2991. We conclude that the advantages gained through the use of each machine should be evaluated within the context of the specific intention for the graft. Future advances in the identification and understanding of the primitive stem cell will aid in attempts to evaluate the methods used to isolate these cells.


A morphometric analysis of trimethyltin-induced change in rat brain using the Timm technique.

Rats were given a single gavage of trimethyltin chloride (TMT) providing a dose of 0, 4.3, or 6.7 mg/kg of alkyltin. Gross changes in brain structures were quantified and analyzed statistically. Behavioral and functional measures were taken to verify efficacy of TMT dose. The high dose produced transient weight loss and seizures. In the fourth week after gavage, the high dose produced hyperactivity in the residential maze and activity wheel. High and low TMT doses decreased auditory startle responsiveness. Estrus cycle was normal in all groups. Brains were sectioned and stained with the Timm stain which delimited subregions of hippocampus and connected structures and also revealed mossy fibers. Linear and areal measures were made at three positions along the septotemporal axis of Ammon's horn. The low dose produced reductions in size in a few isolated subareas of the brain. The high dose produced, at the three planes studied, extensive (15-40%) loss of tissue in Ammon's horn and structures to which Ammon's horn is interconnected--subiculum, entorhinal cortex, dentate gyrus, hilus, CA3, and CA1 region. Neocortex and caudate-putamen were unaffected. These findings suggest that a single TMT gavage may disrupt brain structures important to linking neocortex with subcortex via structures in the hippocampal region.


Substance P synaptic interactions with GABAergic and dopaminergic neurons in rat substantia nigra: an ultrastructural double-labeling immunocytochemical study.

The distribution of substance P (SP), tyrosine hydroxylase (TH), and glutamic acid decarboxylase (GAD) immunoreactivity in the substantia nigra of the rat was studied by means of an ultrastructural double-labeling immunocytochemical method. Direct synaptic contact between SP-immunoreactive terminals and GAD-positive nigral neurons was more often observed in the pars lateralis than the pars reticularis and was rarely observed in the pars compacta. Substance P-positive terminals also formed synapses with cell bodies and dendrites of TH-positive, dopaminergic neurons in the pars compacta and pars reticulata. Multiple SP-immunoreactive terminals were often observed with symmetrical and, less frequently, asymmetrical synapses on individual TH-containing dendrites. Evidence of SP-containing terminals contacting both GABAergic and dopaminergic neurons in the substantia nigra suggests a direct excitatory action upon nigral projection neurons.


Neuropeptides in the torus semicircularis of the carp (Cyprinus carpio).

The distribution of fibers and cell bodies containing neurotensin, neurokinin A, galanin, or somatostatin-28(1-12) immunoreactivity in the torus semicircularis of the carp was studied using an indirect immunoperoxidase technique. In this mesencephalic region, a high-density of galanin-immunoreactive fibers was found, whereas neurokinin A or somatostatin-28(1-12)-immunoreactive processes were observed at a moderate density and neurotensin-immunoreactive fibers at a low density. Cell bodies containing somatostatin-28(1-12) immunoreactivity were observed in both central and lateral nuclei. The torus semicircularis was not immunoreactive for dynorphin A. The presence of these neuropeptides in the carp torus semicircularis suggests that such neuroactive substances may be involved in auditory and visual mechanisms, as well as in the control of inputs arising from the lateral line system.


Efferent projections of the nucleus raphe magnus.

This study was performed to identify supraspinal efferents of the nucleus raphe magnus (NRM) in the rat using the anterograde tracer phaseolus vulgaris leucoagglutinin (PHA-L). NRM-derived PHA-L-ir fibers, with putative terminals, were identified in regions including the lateral hypothalamus, parafascicular nucleus, ventral lateral periaqueductal gray (PAG), locus coeruleus, parabrachial nucleus, A7, A5, and nucleus tractus solitarius. Projections to the PAG demonstrate reciprocity in PAG-NRM connectivity that may modulate the PAG-NRM-spinal cord pathway. The NRM may contribute to supraspinal modulation of nociception by efferents identified in the PAG, as well as locus coeruleus, A7, and A5, which have been shown to project to the spinal cord dorsal horn. Our results provide neuroanatomical support for NRM involvement in supraspinal mechanism(s) for modulation of nociception.


In vivo measurement of hypothalamic serotonin release by intracerebral microdialysis: significant enhancement by immobilization stress in rats.

Intracerebral microdialysis was used to measure extracellular serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the hypothalamus of unanesthetized rats. Increase in the concentration of K+ in the perfusing Ringer solution (70 mM) produced a sharp increase in serotonin release, which was significantly attenuated by omitting Ca2+ from the perfusion medium. Intraperitoneal injection of 5-hydroxytryptophan, a precursor of serotonin, or local perfusion of pargyline, a monoamine oxidase inhibitor, elevated the hypothalamic serotonin. Releasers or uptake inhibitors of serotonin, such as fenfluramine, cocaine, mazindol, or imipramine, when added to the perfusion medium, significantly increased serotonin level, whereas 5-HIAA was unaffected by these substances. Immobilization-stress caused an immediate increase in both the extracellular serotonin and 5-HIAA in the hypothalamus, suggesting that the hypothalamic serotonergic system is activated during immobilization stress. The present study indicates that the brain microdialysis is useful for analysis of local changes in serotonin concentration which directly reflect neuronal transmission.


The superior cervical ganglion: origin of sympathetic fibers in the facial and hypoglossal nerves in the cat.

Location of superior cervical ganglion (SCG) neurons, sending axons into the facial and hypoglossal nerves, was investigated in the cat by means of retrograde axonal transport of horseradish peroxidase (HRP). After wheat germ agglutinin conjugated HRP (WGA-HRP) was injected into these nerves, many retrogradely labeled neurons were found widely in the ipsilateral SCG, particularly around the caudal half of the SCG. These neurons were round or oval in shape and 70-80% of these were medium in size. In fluorescent experiments, fast blue (FB) was used in combination with diamidino yellow (DY). After injections of FB into the facial nerve and DY into the hypoglossal nerve, a few FB-DY double-labeled neurons occurred in the SCG ipsilaterally.


Progressive accumulation of large aggregates of calcium-containing polysaccharides and basophilic debris within specific thalamic nuclei after lithium/pilocarpine-induced seizures.

Between 30 and 50 days after the induction of seizures by a single injection of lithium and pilocarpine, large aggregates of Nissl-staining material appeared; they occupied up to 35% of the thalamic volume. Both histochemical and atomic absorption analyses indicated elevated concentrations of Ca++ (and possibly Mg++) within this substance that was also composed of polysaccharides and nucleic acids. Significant interactions between time since seizure induction and form of the material indicated a progressive accretion of this material from diffusely scattered micrometer granules to large crystalline forms. We suggest this material is composed of endoplasmic reticular debris that is bound by bivalent cations; because the severity of damage exceeds local phagocytic capacity, the material aggregates and then crystallizes. Possible relation to thalamic calcification in neonatal ischemic brains is considered.


[Prospective study on the diagnosis of hepatocellular carcinoma by using alpha-fetoprotein reactive to lentil lectin].

Levels of alpha-fetoprotein reactive to lentil lectin (AFP-R-L) were monitored in 27 patients with 20-400 micrograms AFP/L and negative imaging. These cases finally were proven to be affected by hepatocellular carcinoma. AFP-R-L above 25% was used to define the positive result for hepatocellular carcinoma. The positivity of AFP-R-L was 63%. At the same time, AFP-R-L was detected in 71 cases of chronic liver diseases. All but one had negative AFP-R-L values. The results suggested that AFP-R-L could make a dissection 3-28 months earlier than positive imaging. An accuracy of predicting hepatoma with positive AFP-R-L was 94%. So the diagnosis of hepatoma with negative imaging could be early made by measurement of AFP-R-L.


Intracellular kinetics of the activator calcium of rat heart after ischemic arrest and cardioplegia: quantitative comparison of right and left ventricles.

The effects of plain ischemia (34 degrees C) and the protective role of hypothermia (20 degrees C) alone or in combination with cardioplegia (St Thomas' Hospital [STH] or glucose-potassium-nifedipine [GPN]) on the intracellular kinetics of the activator calcium of cardiac muscle were quantified and compared from the interval-force behaviour (mechanical restitution) of right and left ventricles of the perfused rat heart. Plain ischemia caused a major depression in the restitution of force of contraction of both ventricles, deranged the mixed linear-exponential functions by significantly increasing the time constants of the fitted mechanical restitution curves (MRC) and altered the control right/left ventricle interval-force relationship. The right ventricle was found to be more susceptible to ischemic damage than the left ventricle, and its inotropic reserve was virtually abolished by 1 h of plain ischemia. Hypothermic preservation during ischemia improved the mechanical restitution, salvaged the inotropic reserve and optimized right/left ventricle interval-force relationship, but the time constants of the fitted MRCs were still prolonged. However, both the cardioplegic formulations were equally effective in normalizing the time constants of the fitted curves. In general, right ventricle functions were better preserved by STH cardioplegia and left ventricle functions were better preserved by GPN cardioplegia. Cardioplegic interventions did not further improve the ventricular inotropic reserve compared with hypothermic preservation. Additional beneficial effects of cardioplegic formulations were directed towards stabilizing the linear-exponential functions and hence restitution of force of contraction.(ABSTRACT TRUNCATED AT 250 WORDS)


A prospective assessment of quality of life after endoscopic intubation and laser therapy for malignant dysphagia.

This study evaluated the effect of endoscopic treatment for malignant dysphagia on quality of life (QL) as part of a prospective comparison of Nd:YAG laser therapy and intubation. Two QL instruments were used: the Quality of Life Index (QLI) and a Linear Analogue Self-Assessment (LASA). Only 23 of 43 patients receiving laser therapy and 15 of 30 having endoscopic intubation agreed to partake in QL assessment; serial measurements until death were obtained in 13 and 9 patients, respectively. Dysphagia grade (DG) as measured on a 5-point scale, correlated significantly with LASA (n = 92; r = -0.51; P less than 0.0001) and QLI (n = 92; r = -0.43; P less than 0.0001) scores. In addition, there was a strong correlation between LASA and QLI scores (r = 0.678; P less than 0.0001). All patients followed up serially until death derived significant palliation of their dysphagia with laser treatment and intubation. Such therapy resulted in a significant initial improvement in QL, with the mean best LASA and QLI scores after treatment being higher than the corresponding mean pretreatment scores (P less than 0.004). However, this improvement proved transient; QL worsened significantly as a patient's general condition deteriorated during the final stages of the illness. The mean last post-treatment LASA and QLI scores in both groups (recorded within 5 weeks of death) were less than the corresponding mean pretreatment scores (P less than 0.004). Endoscopic palliation of malignant dysphagia results in a significant initial improvement in QL. Subsequently, QL worsens appreciably as a patient's general condition deteriorates during the terminal phase of disease.


Periodic acid thiosemicarbazide gelatin methenamine silver (PATSC-GMS) staining for transmission electron microscopy.

A new electron microscopic method is described in which superior staining of basement membranes, collagen fibers and glycogen particles in various tissues was achieved. This method uses a gelatin methenamine silver solution applied to the ultrathin sections on nickel grids after oxidation in a periodic acid. The staining results were excellent and tissue components were evenly stained with no background silver grains. This method proved to be consistent, reproducible and reliable.


Inhibition of murine tumor growth by an interferon-inducing imidazoquinolinamine.

The low-molecular-weight imidazoquinolinamine derivative, 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine (imiquimod, previously described as R-837), induced alpha-interferon (IFN-alpha) in mice. IFN induction was identified at oral doses as low as 3 mg/kg. The 10% lethal dose for daily treatment with imiquimod was 200 mg/kg. Oral treatment with 30 mg/kg imiquimod once every three days significantly inhibited MC-26 colon carcinoma. Delay of treatment from day 1 to day 5, when tumors were easily palpable, did not reduce benefits. Ten daily treatments were slightly more effective than five. However, delivery of the same total dose of imiquimod either once every day for 20 days, once every 4 days, once every 7 days, or once every 10 days inhibited tumor growth to the same level. The antitumor effects of imiquimod were significantly abrogated by an antiserum to murine IFN-alpha, suggesting that the antitumor effect was to a substantial extent mediated by IFN induction. Imiquimod also significantly reduced the number of lung colonies in mice inoculated i.v. with MC-26 tumor cells. Combination of treatment with imiquimod and cyclophosphamide was significantly (P less than 0.01) better than treatment with either drug alone. Combination treatment with cyclophosphamide led to cures in some of the mice inoculated either s.c. or i.v. with MC-26 cells. Treatment with imiquimod also inhibited the growth of RIF-1 sarcoma and Lewis lung carcinoma but was ineffective for P388 leukemia. Imiquimod is an oral IFN-alpha inducer with antitumor effectiveness for transplantable murine tumors.


Angiogenesis in atherosclerosis.

This article reviews studies on the structure, complications and pathogenesis of angiogenesis in atherosclerotic plaques. Although important, this topic has not been studied extensively; the authors present a review of the important information available in the literature. Since angiogenesis has profound clinical complications, the focus is on data which provided information to understand the nature of the vascular structures involved and the sequence of events which led to the initiation and growth of these small vessels in the plaque. This review indicates that it is most likely that the capillaries that first form arise from the vasa vasorum. Since the cell biology literature suggests that angiogenesis is regulated by angiogenic factors which stimulate vascular cell migration and proliferation, the known role of several growth factors and inhibitors is reviewed. It is likely that a complex interaction in the atherosclerotic vessel wall results in angiogenesis, and that further study with purified growth factors and other substances is needed to clarify the pathogenesis of this important biological process.


Expression of human hsp70 in rat fibroblasts enhances cell survival and facilitates recovery from translational and transcriptional inhibition following heat shock.

We report here our studies on the inhibitional effect of 45 degrees C heat shock on transcriptional and translational activity and their subsequent recovery at 37 degrees C in Rat-1 cells, thermotolerant Rat-1 cells (TT Rat-1), and Rat-1 cells transfected with human hsp70 gene (HR24, M21). Specifically, we ask whether overexpression of hsp70 protects cells from heat-induced inhibition in RNA and protein synthesis, and/or facilitates cells' ability to recover from translational or transcriptional inhibition after heat shock treatment. Our data demonstrate that the constitutive expression of human hsp70, by itself, confers thermal resistance as expressed in enhanced survival and translational tolerance, but not transcriptional tolerance. In addition, the expression of human hsp70 in Rat-1 cells facilitates cells' ability to recover from heat-induced inhibition in protein and RNA synthesis. After healing at 45 degrees C for 25 min, the time required for RNA and protein synthesis to recover is considerably shorter in HR24, M21, and TT Rat-1 cells than that in control Rat-1 cells. These results provide strong evidence for a direct link between the expression of a functional form of mammalian hsp70, and cells' translational tolerance, as well as their ability to recover from translational and transcriptional inhibition after heat shock.


Differentiation of HT-29 human colonic adenocarcinoma cells correlates with increased expression of mitochondrial RNA: effects of trehalose on cell growth and maturation.

The HT-29 human adenocarcinoma cell line has been used extensively in the study of colonic cell differentiation and colon cancer. We report here that substitution of glucose with trehalose (alpha-D-glucopyranosyl-alpha-D-glucopyranoside) depresses growth and promotes mucin-producing, goblet-like maturation of HT-29. An initial characterization of this process was made by analyzing several cDNA clones whose RNA templates were differentially expressed at elevated levels in cells grown in trehalose-containing medium. Seven of the 9 clones examined corresponded to 6 mitochondrial genes whose expression levels, relative to those from glucose-grown cells, ranged from approximately 3-fold for 16S rRNA to 8-23-fold for NADH dehydrogenase subunit 4. On the other hand, levels of mitochondrial DNA copy, measured by using NADH dehydrogenase subunit 4 cDNA as probe, were shown to be unaffected by trehalose treatment. Elevation of cellular NADH dehydrogenase subunit 4 RNA in HT-29 cultures grown in medium containing different components (sodium butyrate, galactose, no-sugar, glucose, cellobiose) generally correlated with depressed growth levels and specifically with increased numbers of mucin-producing cells present. Like butyrate, the sugar, trehalose, is an effective inducer of HT-29 differentiation, and may prove useful as a dietary therapeutic, and as a probe for elucidating mitochondrial involvement in colonic cell differentiation and transformation.


Specificity and immunobiological properties of monoclonal antibody IMH2, established after immunization with Le(b)/Le(a) glycosphingolipid, a novel extended type 1 chain antigen.

Extended lacto-series type 1 chain antigens lacking type 2 chain core have recently been shown to comprise a new type of tumor-associated carbohydrate antigen. Examples are Le(a)/Le(a) (IV3Gal beta 1----3[Fuc alpha 1----4]Glc-NAcLc4Cer) and Le(b)/Le(a) (IV3Fuc alpha 1----2Gal beta 1----3[Fuc alpha 1----4]Glc-NAcLc4Cer) (M. R. Stroud, et al., J. Biol. Chem., 266: 8439-8446, 1991; Eur. J. Biochem., 203: 577-586, 1992). We have now established an IgG3 mouse monoclonal antibody (IMH2) after immunization of mice with Le(b)/Le(a) antigen; however, monoclonal antibody (MAb) IMH2 reacted not only with the immunogen used but also with Le(y)/Le(x) and to a lesser degree with short-chain Le(y) or Le(b) with hexasaccharide ceramide (i.e., IV2FucIII3FucnLc4Cer or IV2FucIII4FucLc4Cer). It showed a high incidence of staining and strong reactivity with carcinomas of colon, rectum, liver, pancreas, and endometrium, but no reactivity with normal colonic mucosa at various loci, and minimal reactivity with normal liver, pancreas, or uterine endometrium. On the other hand, it reacted with normal gastric mucosa, cecal mucosa, urothelium, adrenal glands, and thymus. Its expression in colorectal tumors and normal cecal tissue was independent of secretor status, whereas that in normal urothelium was dependent on secretor status. MAb IMH2 displayed strong lymphocyte-activated or complement-dependent killing of human colonic cancer Colo205 cells in vitro, and inhibition of Colo205 growth in vivo; this inhibition was comparable to that by MAb NCC-ST-421, which is directed to Le(a)/Le(a) epitope (M. Watanabe, et al., Cancer Res., 51:2199, 1991). These results indicate that a new extended type 1 chain structure, Le(b)/Le(a), is a useful tumor marker associated with carcinomas of colon, rectum, pancreas, liver, and endometrium and that MAb IMH2 has potential diagnostic or therapeutic applicability for these carcinomas.


1-beta-D-arabinofuranosylcytosine-diphosphate-choline is formed by the reversal of cholinephosphotransferase and not via cytidylyltransferase.

In an effort to identify the pathway leading to the formation of 1-beta-D-arabinofuranosylcytosine-diphosphate (ara-CDP)-choline from 1-beta-D-arabinofuranosylcytosine (ara-C) treatment of cultured cells, as well as of cells obtained from leukemia patients, we probed the enzymatic steps involved in the CDP-choline pathway for phosphatidylcholine biosynthesis. Ara-C-triphosphate was not a substrate for CTP:phosphocholine cytidylyltransferase activity under the conditions employed, whereas CTP and dCTP were utilized to form CDP-choline and dCDP-choline, respectively. When presented together, ara-C-triphosphate and CTP inhibited the enzymatic conversion of CTP to CDP-choline in the presence of phosphocholine, with a Ki of 6 mM. Since CTP:phosphocholine cytidylyltransferase did not appear to be responsible for the increased levels of ara-CDP-choline, we next studied the other enzyme in the pathway for phosphatidylcholine synthesis that could form ara-CDP-choline, CDP-choline:1,2-diacylglycerol cholinephosphotransferase. CDP-choline:1,2-diacylglycerol cholinephosphotransferase activity present in microsomes isolated from L5178Y murine leukemia cells exhibited a reversal of its normal catalytic activity, using CMP and 1-beta-D-arabinofuranosylcytosine-monophosphate (ara-CMP) along with phosphatidylcholine to produce either CDP-choline or ara-CDP-choline, plus diradylglycerol. The Vmax and Km values for CMP were 0.78 +/- 0.04 nmol/min/mg and 340 +/- 20 microM, respectively, whereas the Vmax and Km for ara-CMP were 0.22 +/- 0.06 nmol/min/mg and 1410 +/- 540 microM, respectively. A Ki value of 3 mM was obtained for ara-CMP under the cell-free assay conditions used. These results indicate that ara-CDP-choline most likely arises from a reversal of the CDP-choline:1,2-diacylglycerol cholinephosphotransferase utilizing ara-CMP, rather than from the catalysis of ara-C-triphosphate plus phosphocholine to ara-CDP-choline by CTP:phosphocholine cytidylyltransferase. It is speculated that this mechanism may explain, in part, the rapid cellular lysis observed with high dose ara-C therapy.


Free human chorionic gonadotropin beta subunit in gonadal and nongonadal neoplasms.

The diagnostic value of elevated human chorionic gonadotropin (hCG) and its free alpha (hCG alpha) and beta (hCG beta) subunit serum levels as specific tumor markers for nongonadal malignancies is controversial. In the present report, different monoclonal based immunoradiometric assays specific for hCG and its free hCG alpha and hCG beta subunits have been used to reevaluate the presence of these molecules in the serum of patients with a wide variety of tumors. Serum samples from patients with newly diagnosed, persistent, or recurrent malignancies of either known (n = 717) or unknown (n = 32) primary site, healthy blood donors (n = 309), and nonmalignant disease controls (n = 86) were studied using four highly specific and sensitive monoclonal based immunoradiometric assays to hCG and its free subunits. Low level hCG elevations (less than 1000 pg/ml) were found to be common in cancer patients, normal subjects, and disease controls. However, serum levels greater than 1000 pg/ml were highly diagnostic of gonadal tumors and specifically identified nonseminomatous testicular tumors. Significant serum elevations of free hCG alpha subunit (as high as 3000 pg/ml) were found in approximately 96% of cancer patients, normal individuals, and disease controls. In contrast, free hCG beta subunit levels (greater than or equal to 100 pg/ml) were detected in 70 and 50% of patients with nonseminomatous and seminomatous testicular cancers, respectively, and in 47% of bladder, 32% of pancreatic, and 30% of cervical carcinomas. All normal subjects and disease controls had free hCG beta levels less than 100 pg/ml. Thus, the detection of the free hCG beta subunit in serum of nonpregnant subjects was highly diagnostic of malignancy in general and specifically defines a subgroup of aggressive nongonadal malignancies.


Intrinsic resistance to methotrexate in human soft tissue sarcoma cell lines.

A human fibrosarcoma cell line, HT-1080, and four new cell lines (HS-16, HS-28, HS-30, and HS-42) were established from untreated patients with mesenchymal chondrosarcoma, peripheral nerve sheath sarcoma, malignant hemangiopericytoma, and mixed mesodermal tumor, respectively, and were used for analysis of mechanisms of intrinsic resistance to methotrexate. All four new cell lines were resistant to methotrexate as determined by inhibition of thymidylate synthase in whole cells and by growth inhibition, as compared with HT-1080, a methotrexate sensitive cell line. Methotrexate uptake, level of dihydrofolate reductase, and inhibition of this enzyme by methotrexate in the four cell lines were comparable to HT-1080 cells. However, levels of long chain polyglutamates (glu3-5) of methotrexate achieved after a 24-h incubation with this drug were much lower in the four new cell lines as compared to the HT-1080 cell line (5- to 20-fold lower). The low levels of methotrexate polyglutamates formed is likely the major cause of intrinsic methotrexate resistance in these new sarcoma cell lines.


HLA class II antigen expression in human papillomavirus-associated cervical cancer.

The observation that tumor cells of some neoplasms display major histocompatibility complex (MHC) class II molecules may be of functional significance, influencing the progression of malignancy by allowing the cancer cells to present antigen to the immune system. In the normal cervix, class II molecules are expressed by columnar but not squamous epithelium. The pattern of MHC class II expression in cervical carcinomas has been documented using immunohistochemical methods. Of 53 cervical squamous carcinomas examined for MHC class II expression, only 17% maintained a negative phenotype characteristic of the epithelium from which they were derived, while the remaining tumors exhibited either uniform (45%) or heterogeneous (38%) expression. Tumor areas which were class II positive also express class II associated invariant chain and the adhesion molecules lymphocyte function antigen 3 and intercellular adhesion molecule 1. The DR, DP, and DQ class II MHC subloci are differentially expressed, suggesting independent regulation. There is a trend for tumors with the uniform class II phenotype to predominantly express DR antigen, whereas tumors of the heterogeneous class II phenotype express with equal frequency either DR or DP antigens dominantly. There is no apparent influence of class II status on lymphocyte infiltration of the tumors. The presence of human papillomavirus 16 DNA in the cervical carcinoma specimens was analyzed by Southern blotting of restriction enzyme digested DNA and no correlation between the presence of human papilloma virus and MHC class II expression was found.


Distribution patterns and coexistence of neurohormonal peptides (ANP, BNP, NPY, SP, CGRP, enkephalins) in chromaffin cells and nerve fibers of the anuran adrenal organ.

Immunohistochemical techniques were used to study the adrenal organs of the anuran species Rana esculenta, Caldula pulchra and Bufo marinus with respect to the distribution and coexistence of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), Leu-enkephalin (Leu-ENK). Met-enkephalin-Arg-Phe (MEAP) and dynorphin A 1-17 (DYN). Antisera against enzymes involved in catecholamine synthesis, i.e., dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylase (TH), were used for the identification of chromaffin cells. ANP-immunoreactive (-IR) cells occurred in high densities (30%-70% of the total cell population) in all species investigated. In C. pulchra and B. marinus, BNP-IR cells constituted a population of non-DBH-IR and non-TH-IR cells that were different from the ANP-IR cells. A large proportion of the adrenal cells (10%-55%) were immunoreactive to Leu-ENK, and a minority (2%-5%) showed MEAP-immunoreactivity. DYN-immunoreactivity was not observed. The anurans studied exhibited small numbers of SP-IR, CGRP-IR and NPY-IR cells. Immunoreactivities for ANP + Leu-ENK and Leu-ENK + MEAP were shown to coexist. In C. pulchra and B. marinus, immunoreactions for ANP + NPY, ANP+SP and SP + CGRP were also colocalized. Except for DYN, all neurohormonal peptides also occurred in intra-adrenal nerve fibers. SP-IR fibers also displayed CGRP-immunoreactivity and some Leu-ENK-IR fibers contained MEAP-immunoreactivity. In C. pulchra, NPY-IR fibers were found that also showed ANP-immunoreactivity.


Immunohistochemical localization of serotonin, leu-enkephalin, tyrosine hydroxylase, and substance P within the visceral sensory area of cartilaginous fish.

We examined the distribution of immunoreactivity to serotonin (5-HT), leu-enkephalin (LENK), tyrosine-hydroxylase (TH), and substance P (SP) within the primary visceral sensory region of cartilaginous fish. Two genera of sharks, Squalus and Heterodontus, a skate, Raja, a ray, Myliobatis, and a holocephalian, Hydrolagus, were used. Cranial nerves, VII, IX, and X enter the visceral sensory complex from the lateral aspect and divide it into lobes. Based on sagittally cut sections, there are four lobes in Hydrolagus and five in Squalus, corresponding to the number of gill arches. The neurochemicals are differentially distributed within each lobe. LENK+ and 5-HT+ fibers are located in all regions within the visceral sensory complex. SP+ fibers are extremely dense in a dorsolateral subdivision and do not extend as far ventrally as 5-HT+ and LENK+ fibers. The lobes lack 5-HT+ cells, but contain a few LENK+ and SP+ cells. Many TH+ cells are distributed in dorsomedial portions of the complex, but there are few TH+ fibers. Thus, the visceral sensory area of cartilaginous fish contains several divisions that can be distinguished by their neurochemical content.


Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis.

Mammalian myogenesis is biphasic: primary myoblasts fuse to form primary myotubes, then secondary myoblasts align along the primary myotubes and form secondary myotubes, which comprise most of adult muscle. We provide evidence that an integrin (VLA-4) and its counter receptor (VCAM-1) have a role in secondary myogenesis. Both receptors are synthesized by cultured muscle cells: VLA-4 is induced as myotubes form, whereas VCAM-1 is present on myoblasts and myotubes. In vivo, both molecules are expressed at sites of secondary myogenesis, VLA-4 on primary and secondary myotubes, and VCAM-1 on secondary myoblasts and on regions of secondary myotubes apposed to primary myotubes. These patterns suggest that VLA-4-VCAM-1 interactions influence alignment of secondary myoblasts along primary myotubes and/or the fusion of secondary myoblasts. In support of the latter possibility, antibodies to VLA-4 or VCAM-1 inhibit myotube formation in culture.


Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases.

The macrolide rapamycin blocks cell cycle progression in yeast and various animal cells by an unknown mechanism. We demonstrate that rapamycin blocks the phosphorylation and activation of the 70 kd S6 protein kinases (pp70S6K) in a variety of animal cells. The structurally related drug FK506 had no effect on pp70S6K activation but at high concentrations reversed the rapamycin-induced block, confirming the requirement for the rapamycin and FK506 receptor, FKBP. Rapamycin also interfered with signaling by these S6 kinases, blocking serum-stimulated S6 phosphorylation and delaying entry of Swiss 3T3 cells into S phase. Neither rapamycin nor FK506 blocked activation of a distinct family of S6 kinases (RSKs) or the MAP kinases. These studies identify a rapamycin-sensitive signaling pathway, argue for a ubiquitous role for FKBPs in signal transduction, indicate that FK506-FKBP-calcineurin complexes do not interfere with pp70S6K signaling, and show that in fibroblasts pp70S6K, not RSK, is the physiological S6 kinase.


[Comparison of ultrastructural and immunohistochemical studies on renal cell carcinoma and human embryo kidney].

26 cases of renal cell carcinoma (RCC) were studied by both light microscopy and electron microscopy, accompanied with 9 cases of human embryo kidney as control. 20 cases of RCC in different degree of cell differentiation, 4 cases of human embryo kidney and 1 case of adult human kidney were studied with immunohistochemical methods. The results indicated that the histostructures and antigen expression of RCC were rather complicated and the ultrastructures and antigen expression changes of RCC seemed very similar to those of the developing embryonic kidneys. This indicated, that RCC might have originated from nephroblastoma cells.


[The changes of substance P contents in central nervous system of renal hypertensive rats].

The changes of substance P-like immunoreactivity (SPLI) were determined in the central nervous system of renal hypertensive rats (produced by application of a constricting clip on the left renal artery), as compared with those of sham-operated controls. The results showed that the substance SP (SP) contents in the rostral ventrolateral medulla (RVM) and intermediolateral cell column (IML) of the thoracic spinal cord in renal hypertensive rats (RHR) were significantly higher than those in the control group. On the other hand, there was no significant difference of SP content in the hypothalamus (HP) between RHR and control groups. Intrathecal injection of SP antagonist lowered the mean blood pressure in both RHR and control groups. This data suggests that the excess SP contents in RVM and IML may be closely related to the pathogenesis of hypertension.


[Effect of hypoxia on vasodilation induced by substance P (SP) and calcitonin gene-related peptide (CGRP)].

In this paper, isolated rabbit arteries (renal artery, RA; femoral artery, FA; basilar artery, BA) were used to study the effect of hypoxia on vasodilation induced by substance P (SP) and calcitonin gene-related peptide (CGRP). The results showed that hypoxia caused a marked decrease of the arteries, response to SP (10(-7) mol/L) and CGRP (2.8 x 10(-9) mol/L). Repeated hypoxia even resulted in a complete disappearance of response. On the second hypoxia, CGRP (2.8 x 10(-9) mol/L) induced vasodilation on BA was vanished. On the third hypoxia, vasodilation induced by CGRP (2.8 x 10(-9) mol/L) on FA and that induced by SP (2 x 10(-7) mol/L) on BA were vanished, and CGRP (2.8 x 10(-9) mol/L) induced a lesser extent of vasodilation on RA. At the fourth time, SP (10(-7) mol/L) -induced vasodilation of FA disappeared, and that on RA was small. At the fifth time, the effect of SP (10(-7) mol/L) on RA was nearly vanished. It is concluded that the effect of hypoxia on CGRP-induced vasodilation is stronger than that on SP-induced vasodilation, and the effect is most pronounced on the BA.


[Effects of substance P in rat spinal cord on the humoral immune response to SRBC].

The effects of substance P (SP) in rat spinal cord on the humoral immune response to sheep red blood cells (SRBC) were investigated by the hemolytic plaque-forming cell (PFC) technique. Radioimmunoassay was used for assessing SP content. Catecholamines contents were measured by high performance liquid chromatography (HPLC) with electrochemical-detection. The results suggest that SP in the spinal dorsal horn, but not in the lateral horn, could inhibit the thymus-dependent humoral immune response to SRBC, and this inhibitory effect might be related to the influence of dorsal horn SP on thymic activity. Increased SP content in the spinal dorsal horn at the peak of a humoral immune response might suppress the immune response and play a negative feedback role, preventing excessive immune response.


[Protective effect of 764-3 on experimental inflammation].

The antiinflammatory effect of 764-3 was investigated in rat models of lung inflammation and pleurisy induced by bleomycin intratracheal instillation and carrageenan intrapleural cavity injection respectively. 4 mg/100 g BW.d 764-3 hypodermal injection significantly inhibited the lung inflammatory changes in bleomycin treated rats. Compared with the experimental group, lung index, histamine content in lung tissue and in bronchoalveolar lavage fluid (BALF), number of PMN in BALF and BALF beta-glucuronidase activity significantly deceased in the therapeutic rats. 8 mg/100 g BW 764-3 intramuscular injection significantly inhibited the volume, number of PMN, protein content and beta-glucuronidase activity increase in carrageenan-induced pleural exudate fluid. The antiinflammatory effect of 8 mg/100 g BW 764-3 was equal to that of 3 mg/100 g BW hydrocortisone. 4 mg/100 g BW 764-3 injection significantly inhibited the increases in PMN numbers and beta-glucuronidase activity in pleural exudate fluid. 2 mg/100 g BW 764-3 injection significantly inhibited the PMN increase in pleural exudate fluid only. 1 mg/100 g BW 764-3 injection showed no antiinflammatory effect. When 764-3 was administered orally, the antiinflammatory effect was unsatisfactory. 5 mg/100 mg BW 764-3 significantly inhibited the volume, number of PMN, protein content and beta-glucuronidase activity of carrageenan-induced pleural exudate fluid. 2.5 mg/100 g BW 764-3 significantly inhibited the volume and protein content increases in pleural exudate fluid. But 3.5 mg/100 g BW 764-3 showed no antiinflammatory effect. The authors indicate that the antiinflammatory effect of orally administered 764-3 was not stable.


Expression of the angiotensinogen gene and localization of its protein in the human heart.

There have been no reports on the presence of the tissue renin-angiotensin system in the human heart, although the presence of angiotensinogen has been described in the animal heart. To determine whether angiotensinogen is synthesized in the human heart, we examined angiotensinogen messenger RNA (mRNA) synthesis in autopsy hearts by using ribonuclease protection assay. As a result, angiotensinogen mRNA was detected in the atrial muscle, muscles of the conduction system, and the left ventricular wall. In the left ventricular wall, mRNA expression was more prominent in the subendocardial muscles than in the midcardial or epicardial muscles. Using a monoclonal antibody to human angiotensinogen in immunoblotting experiments, we detected two closely spaced bands at approximately 70 kd in the heart, which was quite consistent with the human angiotensinogen molecule. Immunohistochemical studies with this monoclonal antibody demonstrated intense immunoreactivity in the atrial muscles, the muscles of the conduction system, and those of the subendocardial layers. We conclude that angiotensinogen was synthesized in the human heart. It was evident that the localization of angiotensinogen was not ubiquitous in the cardiac muscles, showing its predilection for the atrial muscles, muscles of the conduction system, and subendocardial layer of the left ventricle.


Cardiac risk of speed traps.

Twenty-two patients with stable cardiac disease drove into a radar trap while having an ambulatory electrocardiogram. All patients reported cardiac symptoms, heart rate increases, and appearance of repetitive ventricular arrhythmias. Myocardial ischemia was observed in some patients. Being caught while car speeding under the stresses of daily life may induce potentially dangerous cardiac effects. The data confirm the effects of stress and adrenergic tone on ventricular arrhythmias.


Alkaline phosphatase in the lactating bovine mammary gland and the milk fat globule membrane. Release by phosphatidylinositol-specific phospholipase C.

1. Alkaline phosphatase is covalently bound to bovine mammary microsomal membranes and milk fat globule membranes through linkage to phosphatidylinositol as demonstrated by the release of alkaline phosphatase following treatment with phosphatidylinositol-specific phospholipase C. 2. The release of alkaline phosphatase from the pellet to the supernatant was demonstrated by enzyme assays and electrophoresis. 3. Electrophoresis of the solubilized enzymes showed that the alkaline phosphatase of the microsomal membranes contained several isozymes, while only one band with alkaline phosphatase activity was seen in the fat globule membrane. 4. Levamisole and homoarginine were potent inhibitors of the alkaline phosphatase activities in both membrane preparations and in bovine liver alkaline phosphatase, but not in calf intestinal alkaline phosphatase.


Substrate adhesiveness and experimental metastatic potential of rat ascites hepatoma AH7974-derived variant sublines.

Cells of an adherent subline (74AD, adhesion greater than 95%) and a floating subline (74FL, adhesion less than 1%) of rat ascites hepatoma AH7974 produced substrates containing fibronectin (FN), laminin (LN) and type IV collagen (CL-IV), with 74AD cells producing higher levels of each component. 74AD cells possessed high adhesion affinities to LN and CL-IV substrates. By contrast, 74FL cells hardly adhered to these purified attachment proteins. The difference in adhesion between the two lines in vitro tended to increase on incubation of the cells in medium containing fetal bovine serum. However, 74FL and 74AD cells adhered avidly to the extracellular matrix (ECM) of vascular endothelial cells. Although the cell-ECM adhesion apparently was not inhibited by pretreatment of the ECM with anti-FN, anti-LN and anti-CL-IV antibodies, the 74FL cell-ECM adhesion was inhibited considerably by pretreatment of the ECM with a mixture of these antibodies, especially with a combination of anti-FN and anti-LN antibodies. The lung-colonizing potential of 74FL cells was greater than that of 74AD cells, but the liver-colonizing potential of 74FL cells was less than that of 74AD cells. These results suggest that rat ascites hepatoma cells with extremely reduced substrate adhesiveness retain an adhesion mechanism that binds to FN and LN in the ECM of vascular endothelial cells. This mechanism may be a minimum essential unit of tumor cell-ECM adhesion in lung colonization, but the unit is insufficient for liver colonization of these cells.


Determination of the molecular weights of ribonuclease isozymes in a cell-free crude extract of Dictyostelium discoideum, by activity-staining of gels after SDS-PAGE.

1. In a previous report we described three isozymes of intracellular ribonuclease in Dictyostelium discoideum, which were found in vegetative cells. Here we report that the molecular weights of the three isozymes from vegetative cells. 2. They are 14.3 kDa, 60 kDa and 80 kDa, as determined by activity-staining of gels after SDS-PAGE. 3. For renaturation of ribonucleolytic activity from D. discoideum cells after SDS-PAGE, fibrinogen-containing gels were used and gels were washed in aqueous isopropanol to remove detergent. Results of studies by this method suggest that each of these isozymes is composed of only a single polypeptide. 4. The effect of the buffer system on this technique is discussed.


A comparative study of covalently-bound fatty acids in keratinized tissues.

1. Covalently-bound fatty acids were characterized in keratinous tissues obtained from a wide range of animals. 2. 18-Methyleicosanoic acid was a major component in all the mammalian fur samples examined except monotreme fur. In monotreme fur 26-carbon fatty acids predominated. 3. Fatty acids from feather keratin and reptile skin had different profiles to the alpha-keratins of mammalian fur. 4. The major forms of covalently-bound fatty acids are very similar in species that diverged up to 125 million years ago.


The effect of exogenous insulin-like growth factor-I (IGF-I) on the reproductive performance of female rats, and on serum concentrations of endogenous IGF-I and IGF-I binding proteins.

The effect of exogenous IGF-I on the reproductive performance of female rats was examined by infusing either recombinant human IGF-I (400 micrograms/d; n = 19) or vehicle (n = 18) over a four-day period (the time of one reproductive cycle) beginning on the day following estrus. The females were exposed to male rats one day after the infusions had commenced, and were euthanized 15 d later. There was no treatment effect on serum progesterone levels at this time or on the number of fetuses. Furthermore, the number of corpora lutea were not different between the IGF-I and vehicle infused groups (15.8 vs. 14.8; P = 0.09). Total serum IGF-I concentrations, as determined with a polyclonal antiserum based RIA, were increased approximately three-fold in samples obtained 20 hr after commencing the IGF-I infusion. These samples were also analyzed for IGF-I with a monoclonal antibody based RIA previously shown to detect human, but not rat, IGF-I. By subtraction, the concentration of endogenous rat IGF-I was found to be approximately 60% higher in IGF-I-infused rats than in control rats. This increase was likely due to a reduced clearance rate of IGF-I from the circulation, caused by a marked induction of 42-46 kDa and 30-34 kDa IGF-I binding proteins observed in these samples with a ligand blot technique. The binding protein induction indicates that the infused IGF-I was bioactive. This induction may have attenuated the effects of IGF-I on ovarian function.


[Balloon dilatation for prostatic obstruction].

Balloon catheter dilatation of the prostate (BCDP) was performed in 25 patients with prostatic hypertrophy. All patients suffered from dysuria or urinary retention. Follow-up for 8-14 months showed that 20 patients had satisfactory results. The balloon catheter of 25 mm x 50 mm was inflated at 4-6 atm for 10-15 minutes. BCDP was modified for the use in the local hospitals.


Weight loss induced by gastric implant in rats. Effects of capsaicin sensory denervation.

To study the efficacy and mechanism of action of the intragastric bubble, 1- to 5-ml silicone bubbles were surgically implanted into the stomachs of 10- to 12-week-old female rats. To test the hypothesis that the satiety effects of the implant are mediated by visceral sensory nerves, a subgroup was treated as neonates with the sensory neurotoxin capsaicin, 50 mg/kg subcutaneously. In control animals, the implants caused a transient decrease in body weight, compared to sham-implanted animals, most evident at three days and abolished by 18 days after operation. In contrast, capsaicin-treated animals did not lose weight in response to gastric implantation. Substance P was decreased in the vagus nerves of capsaicin-treated animals, confirming sensory denervation. At autopsy, all gastric implanted rats had enlarged stomachs. We conclude that intact sensory innervation is essential for weight loss in response to the gastric bubble.


[Determination of antigen-dependent activity of human lung cancer transfer factor by H3-leucine leucine leukocyte adherence inhibition assay].

This report is to demonstrate the antigen-dependent activity of human lung cancer transfer factor (Sp-TF). Sp-TFM was prepared from spleen of mice immunized with the human lung cancer cell line A549. [3H]-leu leukocyte adherence inhibition assay ([3H]-leu-LAI) was modified to identify activity of Sp-TFM. Leukocytes obtained from non-immunized mice were divided into eight groups as follows: 1. Control without TF or antigen; 2. Sp-TFM and antigen of cell line A549 (A549 Ag); 3. Sp-TFM alone; 4. Sp-TFM and ascitic tumor cell H22 antigen of mice (H22Ag); 5. Sp-TFM and antigen of human gastric cancer cell (HGCCAg); 6. Sp-TFM and antigen of human normal lung tissue (NLTAg); 7. Nor special TF of mice (N-TFM) and A549Ag; 8. A549Ag alone. When normal leukocytes were incubated with Sp-TFM and A549 antigen, the leukocytes adherence inhibition index (LAII) was significantly higher than those of the other groups. The different LAII of Sp-TFM to A549Ag and other experimental groups were highly significant (P less than 0.001). The results demonstrated that Sp-TFM could transfer specific cell mediated immunity to non-immune leukocytes. The TF prepared from spleen of goat immunized with antigen from lung cancer of patients (Sp-TFG) showed antigen specific activity as well as Sp-TFM.


[Agyrophilic nucleolar organizer region counts as related to bromodeoxyuridine incorporation scores in ovarian clear cell carcinoma cells].

The value of agyrophilic nucleolar organizer region (AgNOR) stain as potential technique for the estimation of cell kinetics was assessed in this study. The daily AgNOR counts and the scores of the proliferation-associated marker bromodeoxyuridine (BrdU) in a human ovarian carcinoma cell line-OCC1 for 10 days after subculture were determined. The correlation between AgNOR counts and BrdU incorporation scores was highly significant (r = 0.86, P less than 0.01). Therefore AgNOR counts offer a potential tool to provide cytokinetic information.