Drug-protein binding kinetics in patients with type I diabetes.

Sera from 17 patients with Type I diabetes and 19 healthy volunteers have been examined to evaluate whether the kinetics of the binding of drugs to Site II of serum albumin is altered in diabetes. Stopped-flow measurements showed that the association velocity and the affinity constants of the fluorescent marker dansylsarcosine were significantly lower in diabetes (160 s-1 and 2.0 x 10(5) l.mol-1) than in non-diabetics (196 s-1 and 4.0 x 10(5) l.mol-1). The dissociation velocity was not different [20.3 s-1 vs. 19.4 s-1]. Although patients with a reduced albumin concentration were excluded the diabetics had significantly lower concentrations than the healthy volunteers. There was a significant correlation between decreased glycosylation of albumin and increased association velocity. The dissociation velocity constants were correlated with the molar concentration ratio of free fatty acids/human serum albumin. Thus, the extent of glycosylation and the amount of fatty acids bound per mole albumin can both affect the kinetics of drug binding to Site II. The lower affinity in patients with Type I diabetes is due to the increased in the glycoalbumin concentration.


Interleukin (IL1 to IL7) gene expression in fetal liver and bone marrow stromal clones: cytokine-mediated positive and negative regulation.

Mouse bone marrow and fetal liver stromal clones have been analyzed for their cytokine mRNA expression. The reverse transcriptase polymerase chain reaction (RT-PCR) has allowed us to detect interleukin (IL) mRNA levels, even if synthesized at levels not detectable by Northern blot analysis. We found that stromal cells possess the potential to constitutively express a much larger number of interleukins than previously described. The three stromal clones analyzed here expressed mRNA for IL3 and IL2, in addition to mRNA for IL1, IL4, IL6, and IL7. None of the stromal clones synthesized IL5 mRNA. Cytokine mRNA synthesis by stromal cells was found to be subjected to negative and positive regulation by interleukins. IL2, IL3, IL6, and IL7 gene expression was much more sensitive to cytokine regulation than that of IL1 and IL4.


Tumor necrosis factor alpha in human long-term bone marrow cultures: distinct effects on nonadherent and adherent progenitors.

Although tumor necrosis factor alpha (TNF alpha) exerts a variety of activities on hematopoietic cells, suggesting it may have some potential therapeutic applications, its long-term effects on hematopoiesis are not well defined. Therefore, we took the advantage of long-term bone marrow cultures (LTBMCs) to evaluate the long-term role of TNF alpha on both the microenvironment and the hematopoietic progenitors. LTBMCs were inoculated with 100 U/ml of recombinant human TNF alpha (rhTNF alpha) either at the onset of the cultures (d0) or at day 21 (d21) when the adherent layer (AL) was already established. Then TNF alpha was added at each weekly medium change. The cellularity and the content of progenitors in both the nonadherent layer (NAL) and AL, the formation of the AL, and the presence of various cytokines in the supernatants were examined weekly. The data showed 1) a strong and durable inhibitory effect on total nonadherent cells; 2) a rapid and transient inhibition of NA progenitors, whereas adherent progenitors were lately affected; and 3) microenvironmental changes consisting of the disappearance of adipocytes and the secretion of high levels of interleukin 6. The results suggest that the inhibitory effects of TNF alpha on the NAL are in part counterbalanced by stromal modifications that in turn lead to a faster exhaustion of hematopoiesis.


Aortic features in Tangier disease and pathogenetic considerations--Part I. Fatty dots and streaks.

Morphological studies of aortic fatty dots and streaks, and the adjacent normally appearing intima, in a 5 3/4-year-old boy who died of pneumonia, showed several hitherto unreported features. In lesions, lipid vacuoles and/or other cytoplasmic "inclusions" (ultrastructurally considered to present complex forms of lipids) were present on occasion in the endothelium but consistently involved the (intimal) smooth muscle cells (SMC). Similar changes were present in the adjacent intima, but were here less prominent and "tapered off" distally. A moderate number of macrophages also contained cytoplasmic lipids but such cells entirely free of lipid inclusions were observed, too. Most surprisingly, dilated and many cisternae of rough-surfaced endoplasmic reticulum (ER) in the SMC of lesions were associated spatially with cytoplasmic droplets and other forms of lipids. The results of these studies question the generally accepted central role of macrophages as being primarily involved in the pathogenesis of tissue changes in Tangier disease. It is possible that in view of the absence or paucity of high-density lipoproteins (HDL) and alterations of (their) apo A-I and apo A-II (as well as of other lipids), the arterial SMC may be in some way involved in the metabolism of the above substances in this disorder. Support of this tentative (and highly speculative) assumption must await further work utilizing tissues and cells other than those containing macrophages and other derivatives of reticulo-endothelial system.


Binding of RNA by the alfalfa mosaic virus movement protein is biphasic.

The movement protein of alfalfa mosaic virus was expressed in Escherichia coli and purified by cation exchange chromatography. The purified protein bound single-stranded RNA cooperatively in a biphasic manner. At protein saturation, RNA/protein complexes (designated 'primary complexes') were detected by a nitrocellulose-retention assay within 1 min of mixing, both at 4 and 22 degrees C. In contrast, an incubation of 30 min at 22 degrees C was necessary to obtain electrophoretically retarded complexes ('stabilized complexes'), containing a large number of protein molecules bound stably to each molecule of RNA. Stabilization did not take place at 4 degrees C. The rate of formation of the primary complexes was strongly dependent on protein concentration, and thus appeared limited by a bimolecular interaction. In contrast, the rate of stabilization was independent of protein concentration, suggesting that this process consisted of a rearrangement of the primary complexes without binding of additional protein molecules. In agreement with this suggestion, the amount of complexed RNA at equilibrium was the same when assayed by nitrocellulose retention and by electrophoretic retardation. The possibility that these peculiar kinetics could be caused by the presence of Tween 20 in the incubation media is discussed.


Interleukin-1 beta induces the expression of an isoform of nitric oxide synthase in insulin-producing cells, which is similar to that observed in activated macrophages.

The suppressive and cytotoxic effects of interleukin-1 beta (IL-1 beta) on rodent insulin-producing cells observed in vitro are probably mediated through formation of nitric oxide (NO). In this study we demonstrate that IL-1-induced NO formation in isolated rat islets and insulin-producing HIT cells is more sensitive to inhibition by NG-monomethyl-L-arginine than to inhibition by NG-nitro-L-arginine, thus suggesting that IL-1-exposed insulin-producing cells express an isoform of nitric oxide synthase similar to that present in activated macrophages. Furthermore, IL-1 beta markedly increased the mRNA levels of the inducible macrophage form of nitric oxide synthase in HIT cells.


Mutations in a putative agonist binding region of the AMPA-selective glutamate receptor channel.

The region preceding putative transmembrane segment M1 of the glutamate receptor (GluR) channel is well conserved among subunits and has been proposed to constitute a part of the agonist binding site. The functional significance of this region was examined by introducing point mutations into charged residues of the alpha 1 subunit of the mouse alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective GluR channel. The dose-response relationships of the mutant receptors were studied after expression in Xenopus oocytes by injection of the mutant alpha 1 subunit-specific mRNA together with the wild-type alpha 2-subunit-specific mRNA. Variable changes in the EC50 values for different agonists were found for the replacement of glutamic acid 398 by lysine and for the replacement of lysine 445 by glutamic acid. These residues may be involved in selective interaction of the GluR channel with agonists.


Spontaneous thioester bond formation in alpha 2-macroglobulin, C3 and C4.

Purified alpha 2-macroglobulin and complement proteins C3 and C4 were treated with ammonia to break their intramolecular thioester bonds and reform the original free cysteinyl and glutamyl side chains. When this reaction was performed at low temperature a conformational intermediate was trapped which lacked a thioester, but which could refold to the native structure and spontaneously reform the thioester and full biological function. The findings suggest that these proteins may undergo spontaneous post-translational self-modification forming the thioesters without involvement of enzymes or high energy metabolites such as ATP.


Construction of dystrophin fusion proteins to raise targeted antibodies to different epitopes.

For the study of the structure and function relationship of dystrophin, defective in DMD, and for diagnostic purposes it is important to dispose of antibodies against different parts of the protein. We have made five different constructs for the expression of fusion proteins containing parts of the four domains of dystrophin. Two different recombinant expression vectors, pATH2 and pEX1, were used. Rabbits were immunized with the fusion products and several polyclonal antibodies were raised. At a later stage, monoclonal antibodies were also raised to some of the fusion proteins. One polyclonal antibody, named P20 AB, is directed against the region covering amino acid sequence 1749-2248 or the nucleotide sequence 5456-6953 of the mRNA, which corresponds to the major deletion-prone region of the DMD gene. We show the particular value, sensitivity and specificity of the P20 AB in dystrophin analysis.


Conformation of two non-immunosuppressive FK506 analogs when bound to FKBP by isotope-filtered NMR.

The 3D structure of two unlabeled FK506 analogs, (R)- and (S)-[18-OH]ascomycin, when bound to [U-13C,15N]FKBP were determined by isotope-filtered 2D NMR experiments. The structures for the R and S isomers that bind tightly to FKBP but lack immunosuppressive activity are compared to each other and to the conformation of the potent immunosuppressant, ascomycin, when bound to FKBP. The results are interpreted in terms of calcineurin binding to the FKBP/ascomycin complex.


Activation mechanism of human Hageman factor-plasma kallikrein-kinin system by Vibrio vulnificus metalloprotease.

Vivrio vulnificus, an opportunistic human pathogen, secretes a metalloprotease (VVP). The VVP inoculated into a guinea pig is known to generate bradykinin through activation of the Hageman factor-plasma kallikrein-kinin system. VVP was shown to possess the ability to activate the human system through the same mechanism as that clarified in the guinea pig system, namely, VVP converted both human zymogens (Hageman factor and plasma prekallikrein) to active enzymes (activated Hageman factor and plasma kallikrein), and the then generated kallikrein liberated bradykinin from high-molecular-weight kininogen. However, in the presence of plasma alpha 2-macroglobulin (alpha 2M), the VVP action was drastically decreased. This finding suggests that the human system might be activated only at the interstitial-tissue space which contains negligible amounts of alpha 2M or in the bloodstream of the individuals whose plasma alpha 2M level is extremely reduced.


The molecular mechanism of the control of excitation energy dissipation in chloroplast membranes. Inhibition of delta pH-dependent quenching of chlorophyll fluorescence by dicyclohexylcarbodiimide.

Non-radiative dissipation of absorbed excitation energy in chloroplast membranes is induced in the presence of the trans-thylakoid proton motive force; this dissipation is measured as high energy state quenching of chlorophyll fluorescence, qE. It has been suggested that this results from a low pH-induced structural alteration in the light harvesting complex of photosystem II, LHCII [(1991) FEBS Letters 292, 1-4]. The effect of the carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), on energy dissipation in chloroplast membranes has been investigated. At concentrations below that required to inhibit electron transport, DCCD caused a decrease in the steady state delta pH, completely inhibited qE and also inhibited the low pH-dependent induction of qE. DCCD binding to polypeptides in the 22-28 kDa range correlated with inhibition of qE. It is suggested that DCCD reacts with amino acid residues in LHCII whose protonation is the primary event in the induction of energy dissipation. This LHCII domain may be identical to one forming a proton channel linking the site of PSII-dependent water oxidation to the thylakoid lumen [(1990) Eur. J. Biochem. 193, 731-736].


[Brachytherapy: endobronchial irradiation for primary and secondary bronchial tumors].

Intrabronchial obstruction is one of the serious complications of primary and secondary lung tumors. Brachytherapy (intraluminal irradiation) aims at palliation and opening airway obstruction. Of our first 10 patients, 4 had primary and 6 metastatic pulmonary lesions. Brachytherapy was administered through the fiberoptic bronchoscope under topical anesthesia from a 192iridium source delivered with an afterload system. In 3 patients laser therapy was used initially. In 6 complete opening of the airway was achieved, and in 3 others partial opening. In all patients there was symptomatic relief without significant side-effects. We conclude that endobronchial brachytherapy is a safe, useful procedure for palliation of malignant endobronchial obstruction.


Can somatostatin prevent injection pancreatitis after ERCP?

In a double-blind randomized study, 30 patients received somatostatin infusion during ERCP and 30 patients placebo with the aim of evaluating whether somatostatin can reduce the incidence of injection pancreatitis. S-amylase, U-amylase and S-lipase were evaluated before, during and after (up to 48 hours) ERCP. C-peptide was also determined as a marker of the function of the endocrine pancreas. While no statistically significant effect of somatostatin in terms of amylase and lipase was to be found, somatostatin did significantly decrease c-peptide levels in plasma, indicating that the peptide inhibited beta-cell secretion. About 40% of patients in the somatostatin group and about 50% in the placebo group showed signs of injection pancreatitis (elevated levels of enzymes) and in both groups there are patients with clinically apparent pancreatitis.


Alpha-fetoprotein-producing pancreatic cancer--a case report and review of 28 cases.

The case of a female patient with an alpha-fetoprotein (AFP)-producing acinar cell carcinoma of the pancreas is reported, and 28 cases in the literature are reviewed. In our case, the serum AFP level declined drastically after removal of the tumor, but increased when widespread metastases appeared. AFP was detected in the cytoplasm of the cancer cells by immunohistochemical staining. Immunoelectron microscopic studies revealed AFP on the endoplasmic reticulum of the cancer cells. Of the 28 cases with AFP-producing pancreatic cancer, liver metastases were identified in 21 cases (76% overall). There was no correlation between the serum AFP level and liver metastases. Immunohistochemical studies revealed localization of AFP at the primary lesion in 6 out of eight cases tested. In cases of AFP-producing pancreatic cancer, serum AFP levels are useful for the diagnosis and as a marker for evaluating recurrent disease and therapeutic response, and for the management of gastrointestinal disease it should be remembered that some pancreatic cancers produce AFP.


The degree of variability in the amino terminal region of the E2/NS1 protein of hepatitis C virus correlates with responsiveness to interferon therapy in viremic patients.

We investigated amino acid heterogeneity in the variable regions of the E2/NS1 viral protein in interferon-responsive and interferon-nonresponsive patients with chronic hepatitis C virus infection. The study assessed whether any particular heterogeneity pattern(s) could be useful in predicting responsiveness to interferon treatment. The nucleic acid sequences of the hepatitis C virus genome were analyzed from six patients with chronic hepatitis treated with an interferon-beta, three of whom did not respond to the therapy and another three who showed remarkable improvement in the serum levels of liver enzymes and hepatitis C virus RNA after 6 mo. The complementary DNA clones propagated from each of the nonresponders showed significant diversity of both nucleotide and amino acid sequence, especially at the hypervariable region 1 within the putative E2/NS1 gene of the virus, suggesting that these patients were infected with a large heterogeneous pool of hepatitis C virus variants. In contrast, the responders showed little or no diversity in the sequence of the complementary DNA clones, suggesting that they were infected with one or a small population of viral genotypes containing significantly less variability in the E2/NS1 hypervariable region 1. These results suggested that a large variable population of hepatitis C virus genotypes is implicated in patients who are nonresponders to interferon treatment. In addition, a significant change in the hepatitis C virus genotype population was observed in nonresponders after interferon treatment. This may reflect a differential viral sensitivity to interferon, selective immune pressure by the host or both.


Office aeroallergen sampling.

Aeroallergen sampling is a simple procedure that can be performed in any physician's office. The necessary equipment is a pollen and mold collecting device, a microscope with an eye-piece micrometer, pollen stain, and appropriate slides. The inexperienced observer can learn to count easily recognizable pollen and mold, such as ragweed and Alternaria in a matter of minutes. The counting can be simple (of one or two pollens) and can be complex, including counting of various molds under oil immersion.


Spontaneous exacerbation of disease activity in patients with chronic delta hepatitis infection: the role of hepatitis B, C or D?

Forty-six patients with chronic hepatitis delta virus infection were followed between 6 and 116 mo (mean = 32.8 mo; median = 24 mo). Nineteen patients (41%) demonstrated clinical courses with episodes of biochemical reactivation (ALT levels greater than or equal to 10 times baseline values [group A]). Twenty-seven patients (59%) had stable clinical courses without biochemical reactivation (group B). Patients in group A were younger than those in group B (30.5 vs. 35.3 yr; p = 0.03), were less likely to be intravenous drug abusers (16% vs. 52%; p = 0.01) and were more likely to be homosexual (58% vs. 22%; p = 0.01). Serum hepatitis B virus DNA, hepatitis delta virus RNA, IgM antibody to HBc, HBeAg, antibody to HBe and IgG and IgM antibody to hepatitis delta virus were measured in all patients. In group A, these markers were studied before and during reactivation and during remission. In group B, these parameters were studied in a random fashion at 7- to 10-mo intervals. The presence of antibodies to human immunodeficiency virus and hepatitis C virus was assessed in all patients. A total of 38 biochemical reactivation episodes was noted among the 19 patients in group A. Eleven had sequential changes in hepatitis delta virus markers, suggesting that the exacerbations were due to hepatitis delta virus. In three, the sequential changes of viral markers were consistent with the exacerbations due to hepatitis B virus. In five other patients, no sequential changes in viral markers could be demonstrated to correlate with the biochemical exacerbations.(ABSTRACT TRUNCATED AT 250 WORDS)


Liver/kidney microsome antibody type 1 and hepatitis C virus infection.

Recent studies have shown that hepatitis C virus antibodies are present in a large proportion of patients with autoimmune hepatitis type 2. We have studied 83 patients with liver/kidney microsome antibody-positive type 1 hepatitis. Hepatitis C virus antibodies were sought in every case by second-generation tests (hepatitis C virus enzyme-linked immunosorbent assay and recombinant immunoblot assay). Hepatitis C virus RNA sequences were sought in 22 patients (12 with recombinant immunoblot assay-positive results and 10 with recombinant immunoblot assay-negative results) by means of polymerase chain reaction and by use of primers located in the 5' noncoding region. Sixty-four patients (77%) had positive results for hepatitis C virus antibodies in the enzyme-linked immunosorbent assay test, and 41 (49.3%) were confirmed by recombinant immunoblot assay. Hepatitis C virus RNA sequences were found in all the recombinant immunoblot assay-positive patients but in none of the 10 who were recombinant immunoblot assay-negative. The recombinant immunoblot assay-negative patients were younger than those who were positive (13 +/- 11 vs. 50 +/- 11 years) and had higher gamma-globulin levels and liver/kidney microsome antibody-positive type 1 titers (61% had a titer of 1:1,000 or more, vs. only 17% of the recombinant immunoblot assay-positive patients).(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatitis B and C viral infections in patients with hepatocellular carcinoma.

The prevalence of hepatitis B and C virus infections was studied in 70 patients diagnosed as having hepatocellular carcinoma. In addition to viral serological markers, serum hepatitis B virus DNA and hepatitis C virus RNA were determined with a nested polymerase chain reaction assay. Twelve patients (17%) were HBsAg positive, 26 (37%) had antibodies to HBs, HBc or both and 32 (46%) were negative for all hepatitis B virus serological markers. Prevalence of the antibody to hepatitis C virus was 63% (44 patients). Hepatitis B virus DNA was detected in 24 of the 66 tested patients (36%). Twelve of these hepatitis B virus DNA-positive patients were HBsAg negative (seven were positive for antibody to HBs, antibody to HBc or both and five were negative for all hepatitis B virus serological markers). Hepatitis C virus RNA was found in 42 of 68 patients (62%). A high correlation (95%) existed between hepatitis C virus RNA and hepatitis C virus antibodies. Nevertheless, two patients without antibody to hepatitis C virus had serum hepatitis C virus RNA sequences. Coinfection by the two viruses was detected in nine subjects (14%), but no clinical differences were found between these and the rest of the patients. We conclude that nearly 90% (62 of the 70 patients studied) of cases of hepatocellular carcinoma in our geographical area are related to hepatitis virus infections (detected by serological or molecular studies). Hepatitis C is more prevalent than hepatitis B virus in patients with hepatocellular carcinoma, and the infection is still active when the tumor is diagnosed.(ABSTRACT TRUNCATED AT 250 WORDS)


Cytokeratin expression in mucinous sweat gland carcinomas: an immunohistochemical analysis of four cases.

Four mucinous sweat gland carcinomas were examined for the distribution of cytokeratin (CK) polypeptides using immunohistochemical techniques on paraffin-embedded sections. All the tumour specimens reacted with monoclonal antibodies to CK 7, CK 8, CK 18 and CK 19. Antibodies to CK 1, CK 1/2/10/14, CK 1/5/10/11, CK 13, CK 14 and CK 20 did not stain any of the carcinomas. The results add additional support to the notion that mucinous sweat gland carcinoma represents a tumour histogenetically related to the eccrine secretory coil. Furthermore, the absence of CK 20 might significantly contribute to the differentiation of this tumour from cutaneous metastases from gastrointestinal carcinomas.


Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models.

An efficient method was developed for making complete sets of oligonucleotides of defined length, covalently attached to the surface of a glass plate, by synthesizing them in situ. A device carrying all octapurine sequences was used to explore factors affecting molecular hybridization of the tethered oligonucleotides, to develop computer-aided methods for analyzing the data, and to test the feasibility of using the method for sequence analysis. Further development is needed before the method can be used routinely, but our work shows that it has a number of potential advantages over gel-based methods: it should be easy to automate; the quality of the sequence results can be evaluated statistically; it provides a powerful way of comparing related sequences and detecting mutation; it can be applied to both DNA and RNA; and specific motifs can be incorporated into all sequences of the array to focus analysis on sequences of biological interest.


Mapping the human acetylcholinesterase gene to chromosome 7q22 by fluorescent in situ hybridization coupled with selective PCR amplification from a somatic hybrid cell panel and chromosome-sorted DNA libraries.

To establish the chromosomal location of the human ACHE gene encoding the acetylcholine hydrolyzing enzyme acetylcholinesterase (ACHE, acetylcholine acetylhydrolase, E.C. 3.1.1.7), a human-specific polymerase chain reaction (PCR) procedure that supports the selective amplification of ACHE DNA fragments from human genomic DNA was employed with 19 human-hamster somatic cell hybrids carrying one or more human chromosomes. Informative ACHE-specific PCR fragments were produced from two cell lines, both of which include human chromosome 7, but not with DNA from 17 cell hybrids carrying various combinations of all human chromosomes other than 7. Fluorescent in situ hybridization of biotinylated ACHE DNA with metaphase chromosomes from human peripheral blood lymphocytes revealed prominent labeling on the 7q22 position. Therefore, further tests were performed to confirm the chromosome 7 location. DNA samples from the two cell lines including chromosome 7 and the ACHE gene were positive with PCR primers informative for the human cystic fibrosis CFTR gene, known to reside at the 7q31.1 position, but negative for the ACHE-related butyrylcholinesterase (BCHE, acylcholine acylhydrolase, E.C. 3.1.1.8) gene, mapped at the 3q26-ter position, confirming that these lines contain chromosome 7 but not chromosome 3. In contrast, three other cell lines including chromosome 3, but not 7, were BCHE-positive and ACHE-negative. In addition, genomic DNA from a sorted chromosome 7 library supported the production of ACHE- but not BCHE-specific PCR products, whereas with DNA from a sorted chromosome 3 library, the BCHE but not the ACHE fragment was amplified.(ABSTRACT TRUNCATED AT 250 WORDS)


11p15.5-specific libraries for identification of potential gene sequences involved in Beckwith-Wiedemann syndrome and tumorigenesis.

Constitutional and somatic chromosomal abnormalities of the chromosome 11p15 region are involved in an overgrowth malformation syndrome, the Beckwith-Wiedemann syndrome (BWS), and in several types of associated tumors. The bias in parental origin for the different etiologic forms of this syndrome and for loss of heterozygosity in the tumors suggests that a gene (or genes) mapping to this region undergoes genomic imprinting. However, the precise localization of the locus (or loci) for the BWS and associated tumors is still unknown and more markers are required. We therefore isolated 11p15 markers from two libraries: the first one obtained by microdissection of the chromosome 11p15.5 region and the second one, a phage library, constructed from a hybrid cell line containing this region as its sole human DNA. Of 19 microclones isolated from the microdissection library, 11 were evolutionarily conserved. Four phage clones were isolated; one (D11S774) detected a highly informative variable number of tandem repeats (VNTR) and another (D11S773) a biallelic polymorphism. These clones were sublocalized using a panel of somatic cell hybrids that defines eight physical intervals in 11p15.5. Twenty-one clones map to the distal interval that harbors the BWS locus.


PCR amplification and analysis of yeast artificial chromosomes.

A strategy for the analysis of yeast artificial chromosome (YAC) clones that relies on polymerase chain reaction (PCR) amplification of small restriction fragments from isolated YACs following adapter ligation was developed. Using this method, termed YACadapt, we have amplified several YACs from a human Xq24-qter library and have used the PCR products for physical mapping by somatic cell hybrid deletion analysis and fluorescent in situ hybridization. One YAC, RS46, was mapped to band Xq27.3, near the fragile X mutation. The PCR product is an excellent renewable source of YAC DNA for analyses involving hybridization of YAC inserts to a variety of DNA/RNA sources.


Multiplex PCR amplification of three microsatellites within the CFTR gene.

Multiplex PCR amplification has been developed for three highly polymorphic microsatellites (IVS8CA, IVS17BTA, and IVS17BCA) located in intronic regions of the CFTR (cystic fibrosis (CF) transmembrane conductance regulator) gene. The triplex PCR reaction required different concentrations of each pair of primers and labeling of primers in the same reaction. Total informativity is obtained in 90.25% of couples requiring analysis of polymorphisms, and when triplex microsatellite analysis is combined with analysis for the six most common CF mutations in the Spanish population, informativity reaches more than 99%.


Thermal model for the local microwave hyperthermia treatment of benign prostatic hyperplasia.

A system for the noninvasive localized, hyperthermia treatment of benign prostatic hyperplasia was investigated. The system uses a microwave transrectal antenna with a water cooled jacket to achieve localized hyperthermia. The purpose of this study is to model the temperature rise in the prostate and in the surrounding tissue during treatment. The SAR distribution for the transrectal probe is measured in a muscle tissue equivalent phantom. The SAR information is used with a finite element solution of the bioheat transfer equation to give the temperature rise during the treatment. Also the finite element solution is further used to determine the effect of the microwave power, the cooling fluid temperature and the blood perfusion on the tissue temperature rise. The results of the solution are compared to temperature measurements in a canine protocol. It was found that the maximum temperature rise in the tissue during treatment is 44 degrees C at a depth of 2 cm from the rectal mucosa.


IL-6 and NF-IL6 in acute-phase response and viral infection.

NF-IL6 was originally identified as a DNA-binding protein responsible for IL-1-stimulated IL-6 induction. Direct cloning of NF-IL6 revealed its homology with C/EBP. C/EBP is expressed in liver and adipose tissues and is supposed to regulate several hepatocyte- and adipocyte-specific genes. In contrast, NF-IL6 is suppressed in normal tissues, but is rapidly and drastically induced by LPS or inflammatory cytokines such as IL-1, TNF, and IL-6. NF-IL6 can also bind to the regulatory region of various genes including IL-8, G-CSF, IL-1 and immunoglobulin genes. Furthermore, NF-IL6 is shown to be identical to IL-6DBP, a DNA-binding protein responsible for IL-6-mediated induction in acute-phase proteins, demonstrating that NF-IL6 is responsible for the genes regulated by IL-6. These results indicate that NF-IL6 may be a pleiotropic mediator of many inducible genes involved in acute, immune, and inflammatory responses, like NFkB. In this regard, it is noteworthy that both an NF-IL6 binding site and an NFkB binding site are present in the inducible genes such as IL-6, IL-8, and several acute-phase genes. On the other hand, accumulating evidence has revealed that overproduction of IL-6 may be responsible for the pathogenesis and/or several symptoms of a variety of diseases, including autoimmune diseases, malignancies, and viral diseases. At present, the molecular mechanisms of abnormal expression of the IL-6 gene are not known. Recently it has become evident that interplays between viral proteins and cellular proteins play an important role in viral oncogenesis and infection. The fact that NF-IL6 binds to the enhancer core sequences of various viruses strongly suggests a possible relationship of virus infection and IL-6 expression. In fact some evidence (Mahe et al. 1991, Spergel et al. 1992) indicates that NF-IL6 may interact with viral gene enhancers or viral products, although there are no definite data about the involvement of NF-IL6 in viral pathogenesis. Future studies will be required to clarify whether or not the interplay between NF-IL6 and viral infection is responsible for deregulation of the IL-6 gene.


Indigenous silicone rubber endoprosthesis for malignant esophago-pulmonary fistula.

Endoscopic placement of an esophageal endoprosthesis is the most rational therapy for relieving the distress of malignant esophago-pulmonary fistula. The commercial prostheses are very expensive for widespread use in India. We have indigenously prepared silicone rubber endoprostheses. The wall of the prosthesis was hardened in a graded manner until the desired resistance to compression with flexibility is achieved. This prosthesis was placed successfully in five patients with malignant esophago-pulmonary fistula. Dysphagia and aspiration were relieved in all the patients. One patient had delayed esophageal perforation and died of massive bleeding 3 weeks after the placement of prosthesis. The indigenous endoprosthesis is cost-effective and safe.


Immunoblot analysis of antibody responses to Shigella dysenteriae type 1 infection.

Immunoblot analysis was used to identify the antigenic components of the outer membrane protein (OMP) extract from Sh. dysenteriae type 1 that may be relevant in protection against infection. The OMPs were extracted by ultrasonic disruption followed by Sarkosyl extraction. The macromolecular bands were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), electroeluted to a nitrocellulose matrix and complexed with peroxidase conjugated antibodies in human convalescent sera. IgG specific reaction was found to a major antigenic component at 57 kilodaltons. In addition, weakly reactive three antigenic determinants of greater than 57 kD and four of less than 57 kD were found. Control sera from a rabbit immunized against OMP also exhibited a similar pattern of antigenic reactivity. Some of the OMPs with high antigenicity may be important in immunoprophylaxis.


Methyl parathion induced regional alterations in the regulatory proteins during critical stage of central nervous system development in albino rat pups.

Sublethal doses of methyl parathion (O,O-dimethyl-O-nitrophenyl- thiophosphate) injected intraperitoneally to 15 and 21 day old rat pups induced regional alterations in the central nervous system (CNS) in the levels of total RNA, total proteins, modulatory protein Calmodulin (CaM), in the activity levels of membrane bound enzyme Ca(2+)-ATPase and phospholipids. Levels of RNA and total proteins increased considerably in 15 days old methyl parathion treated (MPT) rat pups. Contrary to this the RNA and total protein content exhibited remarkable decrease in 21 day old methyl parathion treated animals. Calmodulin level showed an increase in cerebral cortex and brain stem and decrease in cerebellum and spinal cord in 15 day old methyl parathion treated rat pups. Whereas the level of Calmodulin decreased in cerebral cortex and cerebellum and increased in brain stem and spinal cord in 21 day old methyl parathion treated rat pups. Activity levels of calcium dependent ATPase showed significant inhibition in all the regions of Central Nervous System (CNS) of 15 and 21 day old methyl parathion treated rat pups. Phospholipids showed a general increase in all the regions of Central Nervous System on methyl parathion exposure. In the light of these observations, it has been suggested that the molecular regulatory mechanisms involving Ca2+/CaM are rendered inefficient due to toxic impact of methyl parathion.


[Current status and trends in treatment of scleroderma].

Owing to the wide variety of symptoms, the long clinical course, the inadequate knowledge of the points at which therapeutic action is appropriate and the difficulty of obtaining objective measurements of the treatment results, therapy for systemic sclerosis has to be planned individually. Besides basic recommendations (avoidance of noxious substances, sensible diet, keeping warm, active exercises), physiotherapy and psychological guidance, the therapy is directed at three pathogenetic complexes. Among the vasoactive substances the prostacyclins, calcium channel blockers and angiotensin-converting-enzyme inhibitors (in the case of complicated renal involvement) are recommended. They inhibit the thrombocyte hyperaggregation and lead to vasodilatation. The anti-inflammatory substances prednisolone and azathioprine also exert immunosuppressant (and cytotoxic) effect. Their use is indicated in inflammatory, immunologically active forms of systemic sclerosis. Antifibrotic agents inhibit cross-link formation, prolylhydroxylase, extrusion of collagen from fibroblasts and, thus, collagen synthesis. In addition, they favour the degradation of collagen via the activation of collagenase. Good results have been reported with penicillamine and penicillin G. Pentoxyphyllin leads to vasodilatation and also inhibits collagen metabolism. Promising agents and procedures for future use include cyclosporin A, CD4 antibodies, photopheresis, interferon gamma and factor XIII. A critical attitude to therapy and a great deal of patience are necessary to avoid harming the patients, especially as it is often some months before any effects of the treatment are seen.


Immunocytochemical localisation of substance P-like nerves in the cardiac ganglia of the monkey (Macaca fascicularis).

Substance P-like immunoreactive (SP-IR) nerves formed 2 types of relationships with nerve cells in the cardiac ganglia of the monkey (Macaca fascicularis). The first type consisted of varicose SP-IR nerve fibres that ramified throughout the cardiac ganglia, forming a loose network with several nerve cell bodies. The second type consisted of several nerve cell bodies enwrapped by a dense pericellular (basket-like) investment of varicose SP-IR nerve fibres. Numerous SP-IR nerve fibres formed perivascular networks around the walls of the blood vessels within the cardiac ganglia and the muscle. The cardiac muscle cells were also innervated either by an isolated single varicose SP-IR nerve fibre or by complex networks. At the ultrastructural level, the substance P reaction product appeared to be associated with the microtubules and the outer mitochondrial membranes of labelled axons. Substance P reaction product was also localised on the membranes of small agranular vesicles of the labelled axon terminals. The SP-IR axons and axon terminals were closely related to the nerve cell bodies and they occurred either singly or in small groups. Most of the axons were enwrapped by a sheath from the adjacent Schwann cells which often exhibited pseudopodia-like processes. None of the axon terminals was observed to make synaptic contact with the cell bodies. However, a few axoaxonal contacts involving SP-IR and non-SP-IR axon terminals were present, the significance of which is not understood. Several axon terminals lay close to blood vessels, and may modulate the activity of these vessels.(ABSTRACT TRUNCATED AT 250 WORDS)


O3-induced airway hyperresponsiveness to noncholinergic system and other stimuli.

The effect of O3 exposure (3 ppm, 1 h) on the in vivo and in vitro airway responsiveness, as well as the changes in cell contents in bronchoalveolar lavage (BAL) fluid, were evaluated 16-18 h after O3 exposure in sensitized and nonsensitized male guinea pigs. The sensitization procedure was performed through repeated inhalation of ovalbumin for 3 wk. Increase in pulmonary insufflation pressure produced by the excitatory nonadrenergic noncholinergic (eNANC) system, histamine, and antigen were assessed in in vivo conditions, whereas airway responsiveness to histamine and substance P was evaluated in in vitro conditions by use of tracheal chains with or without epithelium and lung parenchymal strips. We found that O3 exposure 1) increased the neutrophil content in BAL fluids in both sensitized and nonsensitized guinea pigs, 2) caused hyperresponsiveness to eNANC stimulation in nonsensitized guinea pigs (although combination of sensitization and O3 exposure paradoxically abolished the hyperresponsiveness to eNANC stimulation), 3) increased the in vivo bronchoconstrictor responses to histamine and antigen, 4) caused hyperresponsiveness to substance P in nonsensitized tracheae with or without epithelium and in sensitized tracheae with epithelium, 5) did not modify the responsiveness to histamine in tracheae with or without epithelium (and in addition, epithelium removal caused hyperresponsiveness to histamine even in those tracheae exposed to O3), and 6) produced hyperresponsiveness to histamine in lung parenchymal strips either from sensitized or nonsensitized guinea pigs.


The mitochondrial megachannel is the permeability transition pore.

Single-channel electrophysiological recordings from rat liver mitoplast membranes showed that the 1.3-nS mitochondrial megachannel was activated by Ca++ and inhibited by Mg++, Cyclosporin A, and ADP, probably acting at matrix-side sites. These agents are known to modulate the so-called mitochondrial permeability transition pore (Gunter, T. E., and Pfeiffer, D. R. (1990) Am. J. Physiol. 258, C755-C786) in the same manner. Furthermore, the megachannel is unselective, and the minimum pore size calculated from its conductance is in agreement with independent estimates of the minimum size of the permeabilization pore. The results support the tentative identification of the megachannel with the pore believed to be involved in the permeabilization process.


Voltage activation of heart inner mitochondrial membrane channels.

The patch clamp records obtained from mitoplast membranes prepared in the presence of a calcium chelator generally lack channel activity. However, multiconductance channel (MCC) activity can be induced by membrane potentials above +/- 60 mV [Kinnally et al., Biochem. Biophys. Res. Commun. 176, 1183-1188 (1991)]. Once activated, the MCC activity persists at all voltages. The present report characterizes the activation by voltage of multiconductance channels of rat heart inner mitochondrial membranes using patch-clamping. In some membrane patches, the size of single current transitions progressively increases with time upon application of voltage. The inhibitor cyclosporin has also been found to decrease channel conductance in steps. The results suggest that voltage-induced effects which are inhibited by cyclosporin A are likely to involve either an increase in effective pore diameter or the assembly of low-conductance units. In activated patches, we have found at high membrane potentials (e.g., 130 mV) changes in conductance as high as 5 nS occurring in large steps (up to 2.7 nS). These were generally preceded by a smaller transition. Similar results were obtained less frequently at lower voltages. These results can be explained on the assumption that once assembled the channels may act in unison.


Transmembrane arrangement of mitochondrial porin or voltage-dependent anion channel (VDAC).

Porin or voltage-dependent anion-selective channel (VDAC) is the main protein responsible for the high permeability of the outer mitochondrial membrane. The mitochondrial porin is mainly composed of sided beta-strands, in analogy with bacterial porin, whose structure has been resolved at 1.8 A resolution. In mitochondrial porins the N-terminal region forms an amphipathic alpha-helix, a structure conserved in organisms very distant from the evolutionary point of view. This part of the protein is exposed to the water phase, as demonstrated by ELISA assays. Various extramembranous loops have been identified by specific proteolytic cleavages. These overall, combined results were used to draw a model of the transmembrane arrangement of mammalian porin. This model is compared to other mitochondrial and bacterial porin models.


Determination of the number of polypeptide subunits in a functional VDAC channel from Saccharomyces cerevisiae.

Genes encoding VDAC proteins containing specific site-directed amino acid alterations were introduced into wild-type Saccharomyces cerevisiae. The mutant VDAC proteins form channels with ion selectivities very different from that of the wild-type channel. Therefore, the resulting yeast strains express two different genes capable of coding for functional, yet distinct, VDAC channels. If VDAC were an oligomeric channel, analysis of VDAC from these strains should have revealed not only the presence of channels with wild-type or mutant selectivity but also channels with intermediate selectivities. While channels with wild-type and mutant selectivities were observed with approximately equal frequency, no channels with intermediate selectivity were observed. Sufficient observations were performed with two different mutant genes K61E.K65E and K19E.K61E) that the likelihood of having missed hybrid channels was less than 1 in 10(7). These findings favor the hypothesis that each functional VDAC channel is composed of a single 30-kDa polypeptide chain.


The cation-selective substate of the mitochondrial outer membrane pore: single-channel conductance and influence on intermembrane and peripheral kinases.

The polyanion-induced substate of the outer mitochondrial membrane was studied in vivo and in vitro. Study of the substate in artificial bilayers showed that it is highly cation selective. The induction of the substate in intact mitochondria leads to a complete inhibition of the intermembrane kinases, such as creatine kinase and adenylate kinase, which were excluded from the external ATP pool. Peripheral kinases, such as hexokinase, were blocked when they utilized internal ATP. The results with intact mitochondria suggested the existence of two regions of the outer membrane containing channels of different states, which may be involved in the regulation of intermembrane and peripheral kinases.


A soluble mitochondrial protein increases the voltage dependence of the mitochondrial channel, VDAC.

A soluble protein isolated from mitochondria has been found to modulate the voltage-dependent properties of the mitochondrial outer membrane channel, VDAC. This protein, called the VDAC modulator, was first found in Neurospora crassa and then discovered in species from other eukaryotic kingdoms. The modulator-containing fraction (at a crude protein concentration of 20 micrograms/ml) increases the voltage dependence of VDAC channels over 2-3-fold. At higher protein concentrations (50-100 micrograms/ml), some channels seem to remain in a closed state or be blocked while others display the higher voltage dependence and are able to close at low membrane potentials. By increasing the steepness of the voltage-dependent properties of VDAC channels, this modulator may serve as an amplifier in vivo to increase the sensitivity of the channels in response to changes in the cell's microenvironment, and consequently, regulate the metabolic flux across the outer mitochondrial membrane by controlling the gating of VDAC channels.


Binding of a synthetic targeting peptide to a mitochondrial channel protein.

Membrane crystals of the mitochondrial outer membrane channel VDAC (porin) from Neurospora crassa were incubated with a 20-amino-acid synthetic peptide corresponding to the N-terminal targeting region of subunit IV of cytochrome oxidase. The peptide caused disordering and contraction of the crystal lattice of the membrane arrays. Also, new stain-excluding features were observed on the peptide-treated arrays which most likely correspond to sites at which the peptide accumulates. The stain exclusion zones associated with binding of the targeting peptide (and with binding of apocytochrome c in an earlier study) have been localized on a two-dimensional density map of frozen-hydrated, crystalline VDAC previously obtained by cryo-electron microscopy. The results indicate that both the peptide and cytochrome c bind to protein "arms" which extend laterally between the channel lumens. The finding that imported polypeptides bind to a specific region of the VDAC protein implicates this channel in the process by which precursor proteins are recognized at and translocated across the mitochondrial outer membrane.


The mitochondrial benzodiazepine receptor: evidence for association with the voltage-dependent anion channel (VDAC).

Specific, high-affinity receptors for numerous drugs have recently been localized to mitochondrial membrane proteins. This review discusses the association of the mitochondrial receptor for benzodiazepines (mBzR) with the voltage-dependent anion channel (VDAC), indicating a possible auxiliary role for VDAC as a putative drug binding protein. The proposed subunit composition of the purified mBzR complex isolated from rat kidney mitochondria includes VDAC, which functions as a recognition site for benzodiazepines (e.g., flunitrazepam), the adenine nucleotide carrier (ADC), and an 18 kDa outer membrane protein identified by covalent labelling with the mBzR antagonists isoquinoline carboxamides (e.g., PK14105).


Toward the molecular structure of the mitochondrial channel, VDAC.

A summary is presented of the most recent information about the structure and mechanism of closure of the mitochondrial channel, VDAC. Considerable information has come from studies involving electron microscopy of two-dimensional crystals and from electrophysiological studies of wild-type channels and site-directed mutants. Available evidence points to a beta-barrel as the basic structural model for VDAC. Two models for voltage- or effector- induced closure have been proposed, the first involving removal of strands from the wall of the pore, the second invoking movement of protein domains into the lumen. Experimental strategies to resolve the actual mechanism are presented.


Evidence for extra-mitochondrial localization of the VDAC/porin channel in eucaryotic cells.

The expression of bacterial porin in outer membranes of gram-negative bacteria and of mitochondrial porin or voltage-dependent anion channel (VDAC) in outer mitochondrial membranes (OMM) of eucaryotic cells was demonstrated about 15 years ago. However, the expression of VDAC in the plasmalemma (PLM) of transformed human B lymphoblasts has recently been indicated by cytotoxicity and indirect immunofluorescence studies. New data suggest that the expression of VDAC may be even more widespread. Different cell types express porin channels in their PLM and in intracellular membranes other than OMM. The functional expression of these channels may differ in the various compartments since recent experiments have demonstrated that the voltage dependence and ion selectivity of mitochondrial VDAC may be altered by their interaction with modulators. The present paper proposes a unifying concept for the ion-selective channels of cell membranes, in particular, those whose regulation is affected in cystic fibrosis.


Properties of the inner membrane anion channel in intact mitochondria.

The mitochondrial inner membrane possesses an anion channel (IMAC) which mediates the electrophoretic transport of a wide variety of anions and is believed to be an important component of the volume homeostatic mechanism. IMAC is regulated by matrix Mg2+ (IC50 = 38 microM at pH 7.4) and by matrix H+ (pIC50 = 7.7). Moreover, inhibition by Mg2+ is pH-dependent. IMAC is also reversibly inhibited by many cationic amphiphilic drugs, including propranolol, and irreversibly inhibited by N,N'-dicyclohexylcarbodiimide. Mercurials have two effects on its activity: (1) they increase the IC50 values for Mg2+, H+, and propranolol, and (2) they inhibit transport. The most potent inhibitor of IMAC is tributyltin, which blocks anion uniport in liver mitochondria at about 1 nmol/mg. The inhibitory dose is increased by mercurials; however, this effect appears to be unrelated to the other mercurial effects. IMAC also appears to be present in plant mitochondria; however, it is insensitive to inhibition by Mg2+, mercurials, and N,N'-dicyclohexylcarbodiimide. Some inhibitors of the adenine nucleotide translocase also inhibit IMAC, including Cibacron Blue, agaric acid, and palmitoyl CoA; however, atractyloside has no effect.


Modulation of inner mitochondrial membrane channel activity.

Three classes of inner mitochondrial membrane (IMM) channel activities have been defined by direct measurement of conductance levels in membranes with patch clamp techniques in 150 mM KCl. The "107 pS activity" is slightly anion selective and voltage dependent (open with matrix positive potentials). "Multiple conductance channel" (MCC) activity includes several levels from about 40 to over 1000 pS and can be activated by voltage or Ca2+. MCC may be responsible for the Ca(2+)-induced permeability transition observed with mitochondrial suspensions. A "low conductance channel" (LCC) is activated by alkaline pH and inhibited by Mg2+. LCC has a unit conductance of about 15 pS and may correspond to the inner membrane anion channel, IMAC, which was proposed from the results obtained from suspension studies. All of the IMM channels defined thus far appear to be highly regulated and have a low open probability under physiological conditions. A summary of what is known about IMM channel regulation and pharmacology is presented and possible physiological roles of these channels are discussed.


Inhibition of angiogenesis in vitro and in ovo with an inhibitor of cellular protein kinases, MDL 27032.

Protein kinase C (PKC) was implicated as an important positive regulator of angio-genesis by studies showing that tumor promoting phorbol esters, which activate PKC, stimulate angiogenesis both in vitro and in vivo. Therefore, inhibitors of PKC might be expected to block angiogenesis. MDL 27032 [4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone], an inhibitor of cellular protein kinases, prevented capillary-like tube formation by human umbilical vein endothelial cells (HUVEC) on basement membrane preparations, an in vitro model for angiogenic activity. MDL 27032 had an IC50 = 50 microM, whereas MDL 27044, the 4-methyl analog of MDL 27032, was less effective (IC50 greater than 100 microM). This selectivity was reflected in the relative abilities of the two compounds to inhibit PKC and protein kinase A (PKA) activity prepared from HUVEC, and also to inhibit the basic fibroblast growth factor stimulated proliferation of HUVEC. MDL 27032 (0.3 microgram/egg) also significantly inhibited neovascularization in yolk sac membranes of developing chick embryos, whereas MDL 27044 added at concentrations up to 3 micrograms/egg was not inhibitory when compared with vehicle treated controls. Adhesion of HUVEC to individual extracellular matrix proteins, including laminin, fibronectin, and fibrinogen, but not to the mixture of matrix components or collagen type I and IV, was inhibited after treatment with MDL 27032. These studies suggest that MDL 27032, may have potential as an anti-angiogenic agent because it disrupts both formation of tube-like structures by HUVEC on Matrigel and normal neovascularization in ovo. This inhibition may in part be due to altered cellular interactions with the extracellular matrix.


Role of protein kinase C in transforming growth factor-beta 1 induction of carcinoembryonic antigen in human colon carcinoma cells.

Transforming growth factor-beta 1 (TGF-beta 1) regulates the expression of the carcinoembryonic antigen (CEA) gene family in the human colon carcinoma cell line Moser. The mechanisms through which it acts, however, are unknown. In this communication, several lines of evidence are presented to show that the induction of CEA expression and secretion (collectively called CEA responses) by TGF-beta 1 is associated with protein kinase C (PKC) pathway of signal transduction. Treatment of intact cells with the PKC-specific inhibitor calphostin C down-modulated cellular PKC phosphotransferase activity and blocked the induction of the CEA responses by TGF-beta 1. Depletion of PKC by treatment of intact cells with phorbol ester also blocked the action of TGF-beta 1. The induction of the CEA responses by TGF-beta 1 was also blocked by the protein kinase inhibitor 1-(isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), which also inhibited cellular PKC activity. However, TGF-beta 1 did induce the CEA responses in intact cells treated with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), the calmodulin-dependent phosphodiesterase inhibitor calmidazolium, the diacylglycerol kinase inhibitor R59 022, and the G-protein inhibitors cholera toxin and pertussis toxin. Treatment of intact cells with TGF-beta 1 induced a rapid and transient increase in PKC phosphotransferase activity. TGF-beta 1, however, was unable to induce PKC enzymatic activity in cells pretreated with calphostin C. Therefore, it is concluded that TGF-beta 1 regulates the CEA responses through a signal transducing pathway associated with PKC.


Beta 1 integrins isolated from embryonic chicken fibroblasts bind to monomers and polymers of type I collagen.

The avian integrin beta 1 subfamily consists of multiple alpha-beta subunit heterodimers. We employed two different physical states of type I collagen, monomers and fibrils, in the isolation and characterization of avian collagen integrins. Affinity chromatography showed that three integrins, tentatively designated alpha 155 beta 1 (band 1), alpha 5a beta 1, and alpha 3 beta 1 (band 2), bind fibrillar and monomeric collagen under physiological ionic conditions and require divalent cations for binding activity. Sodium chloride gradients (0-0.5 M) were used to assess the functional ability of the integrins to remain bound to the two forms of type I collagen. The results show that integrins elute from the two forms of collagen with distinct fractionation profiles. One integrin, alpha 155 beta 1, binds fibrillar collagen with relatively higher affinity than the other beta 1 receptors. This same avian integrin, alpha 155 beta 1, is immunoreactive with an antiserum (Hynes et al., 1989) raised against a peptide that corresponds to the entire alpha 5 cytoplasmic domain, and coincidently, part of the alpha 6 cytoplasmic domain (de Curtis et al., 1991). Cell biological studies employing double immunofluorescence show that integrins recognized by this antiserum co-localize with extracellular deposits of type I collagen.


Induction of acetylcholine receptor clustering by native polystyrene beads. Implication of an endogenous muscle-derived signalling system.

Aneural muscle cells in culture often form acetylcholine receptor (AChR) clusters, termed hot spots, which are similar to those found at the postsynaptic membrane both in structure and in molecular composition. Although hot spots form on both dorsal and ventral surfaces of the cell, the ventral ones are better characterized because of their association with sites of cell-substratum contact. To understand the stimuli and mechanisms involved in ventral hot spot formation, native, uncoated polystyrene beads were applied to cultured Xenopus myotomal muscle cells to create local membrane-substratum contacts. These beads were able to induce a postsynaptic-type development as evidenced by the clustering of AChRs and the development of a set of ultrastructural specializations, including membrane infoldings and a basement membrane. Whereas these native beads were effective in inducing clustering, beads coated with bovine serum albumin or treated with serum-containing medium were ineffective. Native beads were also capable of inducing clusters in serum-free medium, indicating that their effect was mediated by endogenous molecules that were locally presented by the beads, rather than by bead adsorption of components in the medium. Heparan sulfate proteoglycan (HSPG) is a major component of the muscle extracellular matrix and our previous study has shown that basic fibroblast growth factor (bFGF), a member of the heparin-binding growth factor (HBGF) family, and its receptor are present in Xenopus myotomal muscle during the period of synaptogenesis. Therefore, we tested the involvement of HBGF in bead induction. The results of this study show the following: (1) preincubation of cultures in heparin, which solubilizes matrix-bound HBGFs, suppressed the bead-induced AChR clustering. (2) Suramin, which interferes with the interaction between several growth factors and their receptors, also inhibited bead-induced clustering. (3) Tyrphostin, which blocks tyrosine kinase activity associated with a number of growth factor receptors, was also inhibitory to the bead effect. (4) The percentage of bead-induced AChR clusters was significantly enhanced by pretreating the cultures with bFGF prior to bead application. This exogenously applied bFGF could be largely removed by treatment of cultures with heparin, suggesting its association with HSPG at the cell surface. (5) An anti-bFGF neutralizing antiserum significantly reduced the efficacy of the bead stimulation. These data suggest that uncoated beads, which adhere to the cell surface and can mimic the cell-substratum interaction, effect a local presentation of HBGFs, such as bFGF, residing with the HSPG to their membrane receptors, thereby locally activating receptor-associated tyrosine kinases.(ABSTRACT TRUNCATED AT 400 WORDS)


Inhibition of nitric oxide synthesis increases focal ischemic infarction in rat.

We investigated whether inhibition of nitric oxide (NO) biosynthesis with N-omega-nitro-L-arginine (NNA), a competitive inhibitor of NO synthase (NOS), would modify the volume of the focal ischemic infarction produced by occlusion of the middle cerebral artery (MCA) in spontaneously hypertensive rats. NNA was infused for 1 h (2.4 mg/kg/h) immediately following occlusion of the MCA. NNA increased lesion volume 24 h later by 32% over controls (150.8 +/- 16.6 to 199.2 +/- 17.4 mm3; p less than 0.001, n = 6). This effect was antagonized by co-infusion of L- but not D-arginine. The antihypertensive rilmenidine (0.75 mg/kg) reduced the lesion by 27% (p less than 0.05, n = 4). Changes in lesion size were confined to the penumbra. NNA increased arterial pressure (AP) (118 +/- 8.9 to 149 +/- 16.0 mm Hg; p less than 0.01, n = 3) but did not change regional CBF. However, elevation of AP did not change the lesion volume or distribution. We conclude that inhibition of the constitutive form of NOS in vivo increases the volume of focal ischemic infarction as a consequence of reduced NO biosynthesis. The absence of NO availability may extend lesion formation by inhibition of reactive hyperemia, platelet disaggregation, and/or release of neuroprotective neuromodulators in the penumbra, which may counteract and override any of its neurotoxic actions.


Area 3a in the cat. I. A reevaluation of its location and architecture on the basis of Nissl, myelin, acetylcholinesterase, and cytochrome oxidase staining.

Current knowledge on the anatomy of area 3a of the cat mainly derives from the cyto- and myeloarchitectonic study of Hassler and Muhs-Clement (J Hirnforsch 6:377, 1964). Previous investigations in the cat had failed to identify a cortical region comparable to monkey's area 3a. In the present study, Nissl, myelin, acetylcholinesterase, and cytochrome oxidase staining techniques were applied to coronal and sagittal serial sections of the cat brain. Area 3a appears as a slender band of cortex between areas 4 and 3b, and in Nissl-stained sections it is mainly characterized by an attenuated granular layer IV, overlying a thin layer V with pyramidal cells of various sizes, including a few large ones. These cytoarchitectonic features are sufficient to differentiate area 3a from neighboring areas, although the borders between them are not sharp in many cases. After the Nissl staining, the acetylcholinesterase staining proved to be the most helpful in defining the structure and borders of area 3a. Acetylcholinesterase staining was dense in layer I (in contrast with a lighter staining of outer layer I in area 4), and light in layers II and IIIa, changing to moderate in IIIc and IV (a pattern which is accentuated in area 3b). Myelin and cytochrome oxidase techniques also yielded differential staining patterns of area 3a and neighboring areas 4 and 3b, although the borders were not easily drawn with these techniques. Whereas our cyto- and myeloarchitectonic findings were comparable to those of Hassler and Muhs-Clement ('64) and applied well to area 3a in the convexity of the hemisphere, we found that most of the area 3a described by these authors in the medial face of the hemisphere had a number of distinguishing architectonic (as well as connectional and physiological) features which enabled us to define it as a separate area (7m). The techniques we used to delineate area 3a are compatible with most current procedures of histo- and immunohistochemical staining of the brain, and may also provide valuable supporting data for electrophysiological studies.


The striatum and the globus pallidus send convergent synaptic inputs onto single cells in the entopeduncular nucleus of the rat: a double anterograde labelling study combined with postembedding immunocytochemistry for GABA.

The entopeduncular nucleus is one of the major output stations of the basal ganglia. In order to better understand the role of this structure in information flow through the basal ganglia, experiments have been performed in the rat to examine the chemical nature, morphology, and synaptology of the projections from the globus pallidus and striatum to the entopeduncular nucleus. In order to examine the morphology and synaptology of pallidoentopeduncular terminals and striatoentopeduncular terminals, rats were subjected to a double anterograde labelling study. The globus pallidus was injected with Phaseolus vulgaris-leucoagglutinin (PHA-L), and on the same side of the brain, the striatum was injected with biocytin. The entopeduncular nuclei of these animals were then examined for anterogradely labelled pallidal and striatal terminals. Rich plexuses of PHA-L-labelled pallidal terminals and biocytin-labelled striatal terminals were identified throughout the entopeduncular nucleus. At the electron microscopic level, the pallidal boutons were classified as two types. The majority (Type 1), were large boutons that formed symmetrical synapses with the dendrites and perikarya of neurones in the entopeduncular nucleus. Type 2 PHA-L-labelled terminals were much rarer, slightly smaller, and formed asymmetrical synapses. It is suggested that the Type 2 boutons are not derived from the globus pallidus but from the subthalamic nucleus. The biocytin-labelled terminals from the striatum had the typical morphological features of striatal terminals and formed symmetrical synapses. The distribution of the postsynaptic targets of the pallidal terminals and the striatal terminals differed in that the pallidal terminals preferentially made synaptic contact with the more proximal regions of the neurones in the entopeduncular nucleus, whereas the striatal terminals were located more distally on the dendritic trees. Examination in the electron microscope of areas where there was an overlap of the two sets of anterogradely labelled boutons revealed that terminals from the globus pallidus and the striatum made convergent synaptic contact with the perikarya and dendrites of individual neurones in the entopeduncular nucleus. In order to examine the chemical nature of the input to the entopeduncular nucleus from the globus pallidus and the striatum, ultrathin sections were immunostained by the postembedding method to reveal endogenous GABA. Three classes of GABA-containing terminals were identified; two of them formed symmetrical synapses and one rare type formed asymmetrical synapses. The combination of the GABA immunocytochemistry and anterograde labelling revealed that both the striatal and pallidal afferents that make symmetrical synapses with neurones in the entopeduncular nucleus, including those involved in convergent inputs, are GABAergic.(ABSTRACT TRUNCATED AT 400 WORDS)


Afferent projections to the cholinergic pedunculopontine tegmental nucleus and adjacent midbrain extrapyramidal area in the albino rat. I. Retrograde tracing studies.

The afferent connections of the pedunculopontine tegmental nucleus (PPT) and the adjacent midbrain extrapyramidal area (MEA) were examined by retrograde tracing with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Major afferents to the PPT originate in the periaqueductal gray, central tegmental field, lateral hypothalamic area, dorsal raphe nucleus, superior colliculus, and pontine and medullary reticular fields. Other putative inputs originate in the paraventricular and preoptic hypothalamic nuclei, the zona incerta, nucleus of the solitary tract, central superior raphe nucleus, substantia innominata, posterior hypothalamic area, and thalamic parafascicular nucleus. The major afferent to the medially adjacent MEA originates in the lateral habenula, while other putative afferents include the perifornical and lateral hypothalamic area, periaqueductal gray, superior colliculus, pontine reticular formation, and dorsal raphe nucleus. MEA inputs from basal ganglia nuclei include moderate projections from the substantia nigra pars reticulata, entopeduncular nucleus, and a small projection from the globus pallidus, but not the subthalamic nucleus. Dense anterograde labeling was observed in the substantia nigra pars compacta, entopeduncular nucleus, subthalamic nucleus, globus pallidus, and caudate-putamen only following WGA-HRP injections involving the MEA. The results of this study demonstrate that the PPT and MEA share many potential afferents. Remarkable differences were found that support distinguishing between these two nuclei in future studies regarding the functional organization of the midbrain and pons. The results, for example, confirm our previous observations that the largely reciprocal connections between the midbrain and basal ganglia distinguish the MEA from the PPT. Afferents from the lateral habenula and contralateral superior colliculus represent extensions of more traditional basal ganglion circuitry which further delineate the MEA from the PPT. The results are discussed with respect to the important role of the midbrain and pons in behavioral state control and locomotor mechanisms.


Pathway from the zona incerta to the superior colliculus in the rat.

In order to test the proposal that the zona incerta contributes to the generation of orienting movements, we examined the synaptic relationships between the incertotectal pathway and the cells of origin of the predorsal bundle. The predorsal bundle cells give rise to the major premotor pathway from the superior colliculus to the brainstem gaze centers. First, cytochrome oxidase histochemistry, gamma-aminobutyric acid (GABA), and glutamic acid decarboxylase (GAD) immunocytochemistry, and the axonal transport of markers were used to define the borders of a ventral subdivision of the zona incerta. This subdivision projects topographically to the same sublamina of the intermediate grey layer of the superior colliculus that contains the vast majority of the predorsal bundle cells. Experiments in which incertotectal cells were labeled by both retrograde transport and immunocytochemistry showed that this pathway is GABAergic. Retrograde and anterograde experiments also showed that this pathway is reciprocated by a pathway from the intermediate grey layer of the superior colliculus to the same ventral subdivision of the zona incerta. Finally, experiments combining axonal transport and electron microscopic methods showed that the incertotectal pathway is the source of a monosynaptic GABAergic input to the cells of origin of the predorsal bundle. The ventral subdivision of the zona incerta is contrasted with a second source of GABAergic input to the predorsal bundle cells, the substantia nigra pars reticulata.


Progressive morphological abnormalities observed in rat spinal motor neurons at extended intervals after axonal regeneration.

It is generally accepted that mammalian spinal motor neurons return to normal after axotomy if their regenerated axons successfully reinnervate appropriate peripheral targets. However, morphological abnormalities, recently observed in spinal motor neurons examined 1 year after nerve crush injury, raise the possibility that delayed perikaryal changes occur after regeneration is complete. In order to distinguish between chronic and progressive alterations in neurons with long-term regenerated axons, rat spinal motor neurons and dorsal root ganglion cells were examined at 5 and 10 months following unilateral sciatic nerve crush. Neurons with regenerated axons were identified by retrograde labelling with horseradish peroxidase. The structural properties of neurons ipsilateral to nerve injury were compared to those of neurons from the spinal cord and dorsal root ganglia on the contralateral side and from age-matched control rats. At 5 months postcrush, the morphology of motor and sensory neurons ipsilateral to injury was comparable to that of control cells. However, several features of the motor neurons with regenerated axons distinguished them from control motor neurons at 10 months postcrush. Mean perikaryal area of ipsilateral spinal motor neurons was larger than the means for control motor neurons (p less than .001). Ipsilateral spinal motor neurons also appeared clustered within the spinal cord and had thicker dendrites. Dorsal root ganglion cells with regenerated axons were slightly larger than control cells at 10 months postcrush but they exhibited no other morphological changes. The present findings indicate that spinal motor neurons are progressively altered after their regenerated axons have reestablished functional synapses with their peripheral targets.


Effect of neonatal degranulation on the morphological development of rat CA3 pyramidal neurons: inductive role of mossy fibers on the formation of thorny excrescences.

Intracellular peroxidase injections were made on CA3 pyramidal neurons from slices obtained from 3-60 day old control and experimental rats. Experimental rats had been irradiated at birth with gamma-rays to destroy the granule cells of the fascia dentata. Neonatal gamma-ray irradiation induced a clear cut reduction of the total number of granule cells (80% decrease at P60) and mossy fibers as revealed by Nissl and Timm stains. The number of branching points and segments, total dendritic length, and density of dendritic spines in the stratum radiatum and stratum oriens on CA3 pyramidal cells were not significantly different between control and irradiated rats. To quantify the development of thorny excrescences we measured the number and total area of the thorny excrescences per neuron at different stages of development. gamma-Ray irradiation induced a reduction in the number and total area of thorny excrescences. This effect was readily apparent by postnatal day (P) 12, and a reduction of 70% in the total area was observed at P30. In conclusion, gamma-ray irradiation destroys the majority of granule cells and induces a reduction in the development of thorny excrescences. Our data strongly suggest that this effect is directly caused by the lack of mossy fibers rather than by the irradiation itself since other parameters (i.e., the number of segments and branching points, density of non-mossy spines, and total dendritic length) were not affected. Therefore, we suggest that mossy fibers play an inductive role in the formation of thorny excrescences.


Ventral pallido-striatal pathway in the rat brain: a light and electron microscopic study.

Most theories of basal ganglia functions have been based on a model circuit in which the flow of information follows a one-way loop proceeding from the cerebral cortex to the striatum, the pallidum/nigra, the thalamus, and then returns to the cortex. However, this model neglects data from several studies that show a direct feedback projection from the pallidum to the striatum. In this study, we have examined this feedback connection in the ventral striopallidal system to determine the morphology and chemical properties of ventral pallido-striatal projection neurons and to determine the morphology of ventral pallidal efferents in the ventral striatum. Fluoro Gold was injected into the ventral striatum to retrogradely label ventral pallidal projection neurons. Substance P immunoreactivity was used as a pallidal marker to delineate the ventral pallidum. The results show that most neurons retrogradely labeled by Fluoro Gold lie in the ventral pallidum. Additional double-labeling experiments show that none of these Fluoro Gold-labeled cells are cholinergic neurons; however, some are immunoreactive for parvalbumin, a calcium-binding protein found in many pallidal neurons. Electron microscopy revealed that the somata and dendrites of these labeled ventral pallidal projection neurons form many synapses with unlabeled terminals. Injection of Phaseolus vulgaris-leucoagglutinin into the ventral pallidum anterogradely labeled many fibers in the ventral striatum. Electron microscopy revealed that these labeled axons form both symmetric and asymmetric synapses with ventral striatal neurons. We have thus confirmed that there is a significant direct projection from the ventral pallidum to the ventral striatum. Whether this projection forms a part of either monosynaptic or polysynaptic feedback loops remains to be clarified. Nevertheless, this pallidostriatal projection must be integrated into the theories on basal ganglia functions.


The influence of inhibitory afferents on the development of postsynaptic dendritic arbors.

The growth and maintenance of dendritic form is dependent on normally functioning excitatory afferents. We have now examined the development of dendritic arbors in the gerbil lateral superior olive (LSO), following contralateral cochlear removal at postnatal day 7, a manipulation that substantially eliminates driven inhibitory transmission. Previous studies have demonstrated that the morphology of LSO dendritic arbors varies with tonotopic position and becomes more restricted with age. The presumed decrease of inhibitory transmission in the contralateral LSO resulted in a hypertrophic response. Quantification of Golgi-impregnated neurons revealed that dendrites had a significantly greater number of branch points, and their arbors were more spread out along the frequency axis compared to normal. This was especially apparent in the high frequency projection region where the glycine receptor density is known to be 4-fold higher than in the low frequency projection region. A measure of LSO nucleus size, cross-sectional area, was identical to control values, indicating no overt signs of degenerative phenomena. Cochlear ablation resulted in a significant atrophy of the ipsilateral LSO, with significant effects on dendritic structure. We conclude that decreased inhibitory transmission during development does not lead to a net degenerative response. Rather, the postsynaptic neurons exhibit a hypertrophic phenotype that may be due to the persistence of an immature state. These results indicate that activity-dependent morphogenetic events are a consequence of both excitatory and inhibitory synaptic transmission.


Transneuronal labeling of cochlear nucleus neurons by HRP-labeled auditory nerve fibers and olivocochlear branches in mice.

Auditory nerve fibers were labeled by extracellular injections of horseradish peroxidase into the spiral ganglion in mice. The labeled fibers were traced in an anterograde direction through the auditory nerve into the cochlear nucleus. In almost half of the injections, the labeled endings of auditory nerve fibers contacted cochlear nucleus neurons that were also labeled with horseradish peroxidase and were presumably transneuronally labeled. Only darkly labeled endings were associated with transneuronally labeled neurons, but not all darkly labeled endings had targets that were transneuronally labeled. Transneuronal labeling occurred almost exclusively in the ventral cochlear nucleus, often between endbulbs and bushy cells. Both "modified" endbulbs and the larger endbulbs of Held transneuronally labeled the bushy cells that they contacted. At the ultrastructural level, transneuronal labeling was evident as a darkening of ribosomes and the membrane surfaces of mitochondria, endoplasmic reticulum, and the nucleus. Transneuronal labeling occurred rarely in octopus, small, and stellate cells, and in neurons of the dorsal cochlear nucleus. Spiral ganglion injections also label olivocochlear fibers, efferent fibers that pass through the ganglion en route to the hair cells. These fibers give off branches to the cochlear nucleus that were rarely associated with transneuronal labeling. In eight instances, the targets of olivocochlear branches were stellate cells or small cells. We suggest that in our mouse preparation, horseradish peroxidase is effective as a transneuronal marker because the short distance from injection site to the cochlear nucleus results in a high concentration of horseradish peroxidase in the endings of the auditory nerve fibers.


Immunolocalization of integrin alpha 6 beta 4 in mouse junctional epithelium suggests an anchoring function to both the internal and the external basal lamina.

The localization of the integrin alpha 6 beta 4, a transmembrane adhesion molecule associated with hemidesmosomes, was studied in mouse junctional epithelium (JE) by the use of monoclonal antibodies in indirect immunofluorescence microscopy. The results showed that the integrin a6 subunit was expressed throughout the JE and was localized to the cell membranes, including the aspects facing the internal and external basal laminae. The beta 4 subunit had a more restricted distribution. It was expressed only in cells facing the internal and the external basal laminae and had a basally polarized distribution. In other parts of gingival epithelium, both integrin subunits were mainly expressed at the basal aspects of basal epithelial cells. The basement membrane components, type IV collagen and laminin, could be detected only in the external basal lamina and in other basement membranes of gingival epithelium. The results indicate that the a6 beta 4 integrin, expressed in mouse JE, has a role in mediating the attachment of the cells to the basement membranes facing the connective tissue and the tooth.


Long-term follow-up of hepatitis B chronic carriers who responded to interferon therapy.

We studied the long-term outcome of patients with chronic hepatitis B virus (HBV) infection who responded to interferon (IFN) therapy. Between 1983 and 1988, 120 patients were included in 5 different protocols; 94 patients were treated with IFN and 26 were controls. Loss of serum HBV-DNA was considered a partial response and occurred in 34 of the treated patients and in 10 of the controls. Only the partial-response patients were followed up for 14-64 months (mean 46 months). HBeAg disappeared in 32/34 of the partial-response treated patients and in 9/10 of the controls. During the follow-up period, 6/34 (18%) treated patients and 1/10 controls suffered a reactivation of the disease with reappearance of HBV-DNA. Only 8/34 (23%) treated patients and 1/10 of the controls lost HBsAg; no statistical differences were observed in baseline characteristics between HBsAg-negative patients and patients who remained HBsAg-positive. Of eight HBsAg-negative treated patients, four were serum HBV-DNA-negative upon polymerase chain reaction and thus formed the HBsAg-negative control cases. Although the frequency of HBsAg loss in treated patients is relatively low, the improvement in liver disease obtained from IFN therapy is sustained over a long period.


Demonstration of peptide-specific and cross-reactive epitopes in proteins reacting with antimitochondrial antibodies of primary biliary cirrhosis.

Recently the main targets of antimitochondrial antibodies (AMA) of primary biliary cirrhosis have been identified as parts of three related mitochondrial multienzyme complexes, namely pyruvate dehydrogenase (PDH), branched chain alpha-ketoacid dehydrogenase (BKDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH). Usually AMA-positive PBC serum samples show reactivity to more than one of these, raising the question whether they are exclusively different antibodies or are, at least in part, the result of cross-reactive specificities. With Western immunoblotting, four antigens with molecular masses of 74, 52, 51 and 43 kDa, are recognized by PBC sera. In this study, using affinity purified antibodies from mitochondrial proteins immobilized on nitrocellulose blots, we demonstrate the presence of peptide-specific and cross-reactive epitopes in some targets. In particular, at least three different epitopes present in the 74-kDa protein (presumed to by PDH-E2) are also present in the 51-kDa protein (probably PDH-X), and two in the 52-kDa peptide (possibly BCKDH-E2). Moreover, the 43-kDa mitochondrial protein (the identity of which is more problematic) has three epitopes. One of these is also present in the 74-, 52- and 51-kDa proteins, a second in the 74- and 51-kDa, and a third seems to be peptide-specific. These results show that different sera with the same immunoblotting pattern of reactivity can have antibodies with different antigenic specificities and, conversely, that the same specificity can be responsible for more than one band.


Post-embedding staining with high-iron diamine-thiocarbohydrazide-silver proteinate and its application to visualizing sulfated glycoconjugates in cryofixed kidney and cartilage.

Cryofixation is generally believed to provide optimal tissue preservation. However, certain post-embedding cytochemical reactions, such as high-iron diamine (HID) staining for sulfated glycoconjugates, are not applicable to cryofixed and freeze-substituted tissues. In the present study, the HID technique was therefore adapted for post-embedding staining. HID staining was performed on thin sections of chemically and cryofixed kidney and growth plate cartilage, embedded in Epon and various acrylic-based resins. All resins and most tissue preparation conditions allowed post-embedding staining with HID, albeit to variable degrees. However, no significant cytochemical reaction was obtained with tissue sections of osmicated kidney embedded in Epon. Profile views of re-embedded sections showed that large stain deposits were usually restricted to the surface, whereas small ones were observed throughout the entire thickness of the section. The staining pattern was essentially similar between chemically fixed and cryofixed specimens. In the glomerulus, stain deposits were mainly seen over the free surface of podocyte foot processes and over the lamina rara externa. The pericellular cartilage matrix of chemically fixed specimens often appeared as condensed elements, usually stained with large deposits. In cryofixed tissues this matrix formed a meshwork composed of thin, extended filamentous structures, many of which showed linear arrays of smaller stain deposits. The data presented here indicate that post-embedding HID-TCH-SP staining can be successfully performed on thin sections of tissues embedded in various resins and, as a result, can be further adapted to cryo-prepared specimens to give a high resolution localization of sulfated glycoconjugates in tissues with optimal molecular preservation.


Differential expression of proline-rich proteins in rabbit salivary glands.

Salivary glands synthesize and secrete an unusual family of proline-rich proteins (PRPs) that can be broadly divided into acidic and basic PRPs. We studied the tissue-specific expression of these proteins in rabbits, using antibodies to rabbit acidic and basic PRPs as well as antibodies and cDNA probes to human PRPs. By immunoblotting, in vitro translation, and Northern blotting, basic PRPs could be readily detected in the parotid gland but were absent in other salivary glands. In contrast, synthesis in vitro of acidic PRPs was detected in parotid, sublingual, and submandibular glands. Ultrastructural localization with immunogold showed heavy labeling with antibodies to acidic PRPs of secretory granules of parotid acinar cells and sublingual serous demilune cells. Less intense labeling occurred in the seromucous acinar cells of the submandibular gland. With antibodies to basic PRPs, the labeling of the parotid gland was similar to that observed with antibodies to acidic PRPs, but there was only weak labeling of granules of a few sublingual demilune cells, and no labeling of the submandibular gland. These results demonstrate a variable pattern of distribution of acidic and basic PRPs in rabbit salivary glands. These animals are therefore well suited for study of differential tissue expression of PRPs.


Plasmodium falciparum-infected erythrocyte receptor(s) for CD36 and thrombospondin are restricted to knobs on the erythrocyte surface.

Adherence of Plasmodium falciparum-infected RBCs (PRBC) to endothelial cells causes PRBC sequestration in cerebral microvessels and is considered to be a major contributor to the pathogenesis of cerebral malaria. Both CD36 and thrombospondin (TSP) are glycoproteins that mediate PRBC adherence to endothelial cells in vitro. Because they are both expressed on the surface of endothelial cells, they probably contribute to PRBC sequestration and vascular occlusion in vivo. By applying affinity labeling of receptor binding sites with purified ligands, we showed for the first time that both CD36 and TSP can bind independently to the PRBC surface and that the PRBC receptor(s) for CD36 and TSP are localized specifically to the electron-dense knob protrusions of the PRBC surface. These findings may help in efforts to develop a malaria vaccine to prevent cerebral malaria.


Superantigen-mediated human monocyte-T lymphocyte interactions are associated with an MHC class II-, TCR/CD3-, and CD4-dependent mobilization of calcium in monocytes.

The present study was designed to examine the potential involvement of calcium ions as second messengers in the mediation of the staphylococcal enterotoxin A (SEA)/MHC class II-induced activation of human monocytes. Treatment of monocytes with a monomeric form of SEA failed to induce detectable changes in the level of intracellular calcium in either monocytes or THP-1 cells. However, cross-linking of SEA with biotin-avidin induced a rapid and transient increase in calcium levels in monocytes and in INF-gamma-treated THP-1 cells. This artificial cross-linking system was reproduced by natural physiologic ligands expressed on the surface of T lymphocytes. Delayed, transient, and concentration (cell as well as toxin)-dependent increases in the cytoplasmic level of free calcium in SEA-treated monocytes were observed upon the addition of autologous resting T cells or purified CD4+ cells, but not of CD8+ cells, B cells, or neutrophils. Antibodies against MHC class II Ag, TCR/CD3, and CD4 molecules inhibited the SEA-dependent interaction between monocytes and T cells as indicated by significant decreases in the rise of calcium levels observed in monocytes. Anti-CD8 and anti-class I antibodies did not affect the interaction between the monocytes and the T cells and failed to alter the calcium response. Taken together, these results suggest that the SEA-induced, T cell-dependent calcium mobilization in monocytes requires physical interactions between SEA-MHC class II, TCR/CD3, and CD4 molecules. The ability to mediate a T cell-dependent calcium increase in monocytes was shared by several enterotoxins including staphylococcal enterotoxin B and toxic shock syndrome toxin-1. The characteristics of the SEA-mediated calcium mobilization in monocytes strongly support the hypothesis that this response is an integral part of the signal transducing machinery linked to MHC class II molecules.


In vivo induced allo-reactive natural killer cells.

We have previously shown that rat allo-selective cells of the CD2+CD5- phenotype were generated in Brown Norway (BN) rats after immunization with allogeneic Wistar/Furth (WF) cells, whereas immunization with semi-allogeneic F1 (WF/BN) cells generated CD2+CD5+ effector T cells. We now report that the allo-selective CD2+CD5- lymphocytes lacked expression of intact CD3 complexes and expressed NKR-P1 molecules although lower as compared to classical NK cells, implicating that these lymphocytes constitute a subset of NK cells. The CD5+ T cells were not cytolytically active in BN rats immunized with WF cells indicating an intersubset regulation with mutually exclusive activation of either allo-selective T cells or allo-selective NK cells. Cold target inhibition showed that lysis of both allogeneic target cells and NK-sensitive target cells was mediated by the same NKR-P1 intermediate effector cells. These NK cells lysed WF but not allogeneic Fischer 344 or autologous BN target cells, indicating selective recognition of an allogeneic determinant. Semiallogeneic F1 (WF/BN) target cells were not lysed. Furthermore, target cells from F1 (WF/BN) x WF back-cross hybrids lacking expression of RT1n (self-MHC class I) were susceptible to lysis, whereas back-cross hybrids expressing RT1n were protected from lysis, indicating that self-MHC molecules conferred protection from lysis. These findings implicate the existence of NKR-P1intermediate and NKR-P1high NK cell subsets with different regulation and function in vivo.


The CD28 ligand, B7, enhances IL-2 production by providing a costimulatory signal to T cells.

Previous studies demonstrated that a human pre-B acute lymphoblastic leukemia cell line, NALM-6, failed to stimulate a primary MLR, despite expression of class II MHC and adhesion molecules. Here we demonstrate that this is the result of the fact that NALM-6 cells do not express the ligand for CD28, namely B7. NALM-6 transfectants that expressed high levels of B7 gained the capacity to stimulate IL-2 production by class II MHC molecule-specific alloreactive T cells and to costimulate a polyclonal population of purified T cells cultured with immobilized anti-CD3 mAb. In the presence of PMA, NALM-6 cells transfected with B7 polyclonally stimulated T cells in a cyclosporine A-resistant fashion, a property previously attributed only to agonistic anti-CD28 mAb. The gain of these functions could not be explained solely by an increased capacity of the transfectants to form conjugates with T cells, suggesting that the CD28/B7 interaction transduces a costimulatory signal in T cells.


Activation-driven T cell death. II. Quantitative differences alone distinguish stimuli triggering nontransformed T cell proliferation or death.

Clonal deletion is the major mechanism by which T cell tolerance is achieved in vivo. The process of activation-driven cell death, originally characterized with T cell hybridomas, likely represents the mechanism of clonal deletion because it shares a number of properties with the in vivo process, especially the ability to be triggered in an Ag-specific manner, the cell-autonomous nature of the response, and its sensitivity to the drug cyclosporin A. We now have extended our analysis of activation-driven cell death to clonal populations of nontransformed T cells. Activation-driven cell death can be induced in nontransformed T lymphocytes by combinations of mitogenic stimuli. In particular, two mitogenic stimuli at high dose, one a lymphokine and the other delivered via the TCR or another activation structure, are required to induce activation-driven cell death. Activation-driven cell death is an active cell suicide process with attributes typical of physiological cell death, including early nuclear disintegration and a requirement for macromolecular synthesis, and is distinct from death by factor deprivation. Susceptibility to the induction of cell death by antigenic or activating stimulation is a common aspect of most T cells and is consistent with observations that clonal deletion can occur throughout T cell ontogeny. Most importantly, the alternative cellular responses of cell death and cell proliferation in nontransformed T cells appear to be triggered solely as a function of quantitative differences in the doses of identical stimuli. This can be viewed as a dose-dependent switch that determines cell fate. Developmental regulation of this switch may explain the processes of positive and negative selection during T cell ontogeny and also provide a mechanistic rationale for a strategy of selective anti-tumor therapy.


A single amino acid mutation in CDR3 of the 3-14-9 L chain abolished expression of the IDA 10-defined idiotope and antigen binding.

The ABPC 48 myeloma protein and the 3-14-9 mAb derive their V region genes from the same VH and V kappa gene families. They also share a cross-reactive idiotope defined by the anti-Id mAb IDA 10. Whereas ABPC 48 is specific for bacterial levan, 3-14-9 showed a significant Ag-binding activity to aminophenyl-beta-N-acetylglucosaminide (AZO). In order to define the molecular basis of idiotope expression and Ag-binding activity, we have cloned the genes encoding the 3-14-9 H and L chain V region genes, generated antibodies that carry mutations within the L chain genes, by site-directed mutagenesis, and investigated the effects of those mutations with respect to IDA 10 idiotope expression and binding to AZO. Our findings show that, whereas expression of the IDA 10-defined idiotope requires association of both the H and L chains, a single change (glycine to phenylalanine) at position 91 in the third complementarity-determining region of the L chain abolished both idiotope expression and Ag-binding activity. In addition, a L chain change of alanine to threonine at position 25 allowed idiotope expression to some extent but significantly reduced binding activity to AZO. These data suggest that a single amino acid change can play a crucial role in the functional activity and structural integrity of antibodies.