Insulin stimulates the tyrosine phosphorylation of a 61-kilodalton protein in rat adipocytes.

Insulin stimulated the tyrosine phosphorylation of a 61-kilodalton (kDa) protein in rat adipocytes prelabeled for 2 h with [32P]orthophosphate. Tyrosine phosphorylation of this 61-kDa protein displayed very similar insulin concentration dependency to receptor autophosphorylation and tyrosine phosphorylation of a high molecular mass receptor substrate of 160 kDa. Phosphorylation of the 61-kDa protein was very rapid with maximum labeling attained at 30 sec, paralleling that of the other two proteins. Phosphoamino acid analysis revealed that each of the insulin-responsive phosphoproteins contained phosphoserine as well as phosphotyrosine, though the ratio of two phosphoamino acids recovered from each protein differed. The 61-kDa protein yielded relatively equal proportions of phosphoserine and phosphotyrosine. In contrast, the insulin receptor yielded relatively more label on phosphotyrosine than phosphoserine, whereas label incorporated into the 160-kDa protein was recovered primarily on phosphoserine. Cleveland peptide maps using either Staphylococcus aureus V8 proteinase or chymotrypsin revealed no similarities between the 61-kDa protein and the other tyrosine phosphorylated proteins. With subcellular fractionation, the 160-kDa protein was found in equal proportions in the high speed pellet (100,000 g) and supernatant. The 61-kDa protein had a similar distribution to that of the 160-kDa protein but was also detected in the low speed pellet (10,000 g). The insulin receptor was localized to the low speed pellet. In summary, rat adipocytes contain an insulin-dependent phosphotyrosyl protein of 61 kDa which is distinct from the more prominent high molecular mass receptor substrate. This 61-kDa protein has characteristics consistent with it being a substrate for the insulin receptor tyrosine kinase.


Effects of tachykinins on luteinizing hormone release in female rats: potent inhibitory action of neuropeptide K.

Tachykinins, a family of biologically active related peptides, are found in variable amounts in the rat hypothalamus. We assessed the effects of five tachykinins, substance P (SP), neurokinin A (NKA), neuropeptide K (NPK), neuropeptide gamma (NP gamma), and neurokinin B (NKB), on LH release in different experimental model systems in ovariectomized rats. In the first series of experiments rats were ovariectomized and implanted with permanent cannulae in the third cerebroventricle of the rat brain. Two weeks later, the effects of intracerebroventricular injection of 0.5 or 1.25 nm various tachykinins on LH release were studied. The results showed that whereas SP, NKA, and NKB were ineffective, and NP gamma was marginally effective, NPK produced a long-lasting suppression of LH release. NPK decreased LH release in a dose- and time-related fashion. Similarly, in the second series of experiments, whereas SP and NKA were inactive, NPK completely suppressed the LH surge induced by progesterone in estrogen-primed ovariectomized rats. In the third series of experiments we observed that NK-2 receptor agonist [Nle10]NKA4-10, and not NK-1 receptor agonist [Sar9,Met(O2)11]SP, suppressed both the release of LH in vivo and basal and KCl-induced hypothalamic LHRH release in vitro. These results show that NPK is the most effective tachykinin in suppressing LH release, and the inhibitory response is mediated by hypothalamic NK-2 receptors. These findings are in accord with the hypothesis that NPK may serve as a hypothalamic inhibitory neurotransmitter/neuromodulator of LHRH secretion.


Expression of messenger RNA for insulin-like growth factors and insulin-like growth factor binding proteins by experimental breast cancer and normal breast tissue in vivo.

We report the expression of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) by breast cancer cells and normal breast tissue in vivo. N-nitrosomethyl-urea (NMU)-induced rat mammary tumors synthesize mRNAs for IGF-II and IGFBP-2, -3, and -4. In contrast, normal lactating breast contains only IGFBP-2 and IGF-II messages; IGFBP-3 and -4 mRNAs are absent in this tissue. IGF-I and IGFBP-1 mRNAs are not expressed in either NMU tumors or in normal breast. This is the first report of in vivo expression of IGFBPs and IGF-II messages in breast tumors.


Dtrk, a Drosophila gene related to the trk family of neurotrophin receptors, encodes a novel class of neural cell adhesion molecule.

We report the identification and molecular characterization of Dtrk, a Drosophila gene encoding a receptor tyrosine kinase highly related to the trk family of mammalian neurotrophin receptors. The product of the Dtrk gene, gp160Dtrk, is dynamically expressed during Drosophila embryogenesis in several areas of the developing nervous system, including neurons and fasciculating axons. gp160Dtrk has structural homology with neural cell adhesion molecules of the immunoglobulin superfamily and promotes cell adhesion in a homophilic, Ca2+ independent manner. More importantly, this adhesion process specifically activates its tyrosine protein kinase activity. These findings suggest that gp160Dtrk represents a new class of neural cell adhesion molecules that may regulate neuronal recognition and axonal guidance during the development of the Drosophila nervous system.


Import of cytochrome c heme lyase into mitochondria: a novel pathway into the intermembrane space.

Cytochrome c heme lyase (CCHL) catalyses the covalent attachment of the heme group to apocytochrome c during its import into mitochondria. The enzyme is membrane-associated and is located within the intermembrane space. The precursor of CCHL synthesized in vitro was efficiently translocated into isolated mitochondria from Neurospora crassa. The imported CCHL, like the native protein, was correctly localized to the intermembrane space, where it was membrane-bound. As with the majority of mitochondrial precursor proteins, CCHL uses the MOM19-GIP receptor complex in the outer membrane for import. In contrast to proteins taking the general import route, CCHL was imported independently of both ATP-hydrolysis and an electrochemical potential as external energy sources. CCHL which lacks a cleavable signal sequence apparently does not traverse the inner membrane to reach the intermembrane space; rather, it translocates through the outer membrane only. Thus, CCHL represents an example of a novel, 'non-conservative' import pathway into the intermembrane space, thereby also showing that the import apparatus in the outer membrane acts separately from the import machinery in the inner membrane.


Expression of factor X and its significance for the determination of paramyxovirus tropism in the chick embryo.

Enveloped animal viruses usually possess a surface glycoprotein which mediates fusion between the viral envelope and host cell membrane, hence enabling the initiation of infection, and its biosynthesis often involves post-translational endoproteolytic activation of the inactive precursor by a host cell protease(s). Therefore, the protease distribution in the host must be critical for determining the viral tropism. We previously isolated from chick embryo a cogent candidate endoprotease of this kind for paramyxovirus infection, and demonstrated its identity with factor X (FX), a vitamin K-dependent serine protease in the prothrombin family which, in general, is synthesized in the liver and circulates as one of the plasma proteases essential for blood clotting. Here, we examined FX expression with specific cDNA and antibody probes in a series of embryonic tissues. Many tissues other than the liver expressed the specific mRNA but, in most instances, the translation products remained inactive zymogen forms. The enzymatically active FXa was detectable only in the allantoic fluid and amniotic fluid, and virus spreading was strictly confined to the tissues in direct contact with these FXa-containing fluids. Thus, the ectopically expressed FXa is probably the major host determinant of paramyxovirus tropism in ovo.


Expression of a constitutive form of calcium/calmodulin dependent protein kinase II leads to arrest of the cell cycle in G2.

Calcium/calmodulin dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase. We have created a calcium/calmodulin independent form of this enzyme by truncation. Expression of this enzyme fragment in a rabbit reticulocyte lysate yields a constitutive enzyme with specific activity similar to the activated native enzyme. We have established mammalian cell lines that transiently express this constitutive enzyme using the glucocorticoid-inducible mouse mammary tumor virus long terminal repeat. The transient increase in kinase activity results in a complete cessation of cell cycle progression. This block develops as a consequence of a specific arrest of the cell cycle in G2. During the block, increases in histone H1 kinase activity present in p13 beads or anti-cdc2 immunoprecipitates are seen in parallel with the accumulation of cells at G2, arguing that the arrest is not due to a failure to activate cdc2 as a histone H1 kinase. These results suggest that other changes in serine/threonine protein phosphorylation besides those involved in activation of cdc2 as a histone H1 kinase may be necessary for proper G2-M transition.


Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism.

Cyclic AMP (cAMP) is the major intracellular second messenger of thyrotropin (TSH) action on thyroid cells. It stimulates growth as well as the function and differentiation of cultured thyrocytes. The adenosine A2 receptor, which activates adenylyl cyclase via coupling to the stimulating G protein (Gs), has been shown to promote constitutive activation of the cAMP cascade when transfected into various cell types. In order to test whether the A2 receptor was able to function similarly in vivo and to investigate the possible consequences of permanent adenylyl cyclase activation in thyroid cells, lines of transgenic mice were generated expressing the canine A2 adenosine receptor under control of the bovine thyroglobulin gene promoter. Thyroid-specific expression of the A2 adenosine receptor transgene promoted gland hyperplasia and severe hyperthyroidism causing premature death of the animals. The resulting goitre represents a model of hyperfunctioning adenomas: it demonstrates that constitutive activation of the cAMP cascade in such differentiated epithelial cells is sufficient to stimulate autonomous and uncontrolled function and growth.


Activation of extracellular signal-regulated kinase, ERK2, by p21ras oncoprotein.

To examine signal transduction events activated by oncogenic p21ras, we have studied kinases that are activated following the scrape loading of p21ras into quiescent cells. We observe rapid activation of 42 kDa and 46 kDa protein kinases. The 42 kDa kinase is the mitogen and extracellular-signal regulated kinase ERK2, (MAP2 kinase), which is activated by phosphorylation on tyrosine and threonine in response to oncogenic p21ras, while the 46 kDa kinase is likely to be another member of the ERK family. Stimulation of these kinases by oncogenic p21ras does not require the presence of growth factors, showing that oncogenic p21ras uncouples kinase activation from external signals. In ras transformed cell lines, these kinases are constitutively activated. We propose that the kinases are important components of the signal transduction pathway activated by p21ras oncoprotein.


Dysregulation of gene expression in mouse trisomy 16, an animal model of Down syndrome.

In humans, trisomy 21 results in a specific phenotype known as Down syndrome (DS). The mechanism by which an extra copy of normal genes leads to the DS phenotype is unknown. Most studies in DS and other aneuploid organisms have shown that gene dose is proportional to gene expression. To date, most genes examined have encoded either metabolic enzymes or constitutively expressed products. In the trisomy 16 mouse, an animal model of DS, we found marked dysregulation of two developmentally regulated genes, App and Prn-p. Dysregulation varied from tissue to tissue and during development in the same tissue. We conclude that abnormal phenotypes seen in aneuploid conditions may result in part from disordered expression of developmentally regulated genes.


Clinical recognition of patients affected by a peroxisomal disorder: a retrospective study in 40 patients.

Peroxisomal disorders are genetic diseases in which an impairment in one or more peroxisomal function(s) causes clinical and multiple biochemical abnormalities. Early recognition of the major peroxisomal disorders in which functional peroxisomes are virtually absent, leading to a generalised impairment of peroxisomal functions, is of utmost importance, as this will enable the prenatal diagnosis of these severe diseases in future pregnancies. Unfortunately, clinical recognition of these disorders can be difficult because of the aspecific and varying phenotypic presentation. We analysed the clinical characteristics in 40 patients suspected of having a peroxisomal disorder to identify specific clinical criteria for diagnosis. From this study we conclude that the combined presence of at least three major clinical characteristics (present in greater than 75% of the affected patients, including psychomotor retardation, hypotonia, impaired hearing, low/broad nasal bridge, abnormal ERG, hepatomegaly) and one or more minor characteristics (present in 50%-75% of the patients, like large fontanelles, shallow orbital ridges, epicanthus, anteverted nostrils, retinitis pigmentosa) warrants biochemical investigation of peroxisomal functions. Further prospective investigations will have to be done to evaluate these criteria.


A major human thyroglobulin epitope defined with monoclonal antibodies is mainly recognized by human autoantibodies.

The antigenic nature of 15 anti-human thyroglobulin (hTg) monoclonal antibody (mAb) epitopes was studied by two different approaches. First, we tested two successive protease-digest products of hTg. Only four mAb from the same cluster of reactivity recognized a low-molecular weight peptide, the other mAb only bound native hTg or high-molecular weight digest fractions. Second, these 15 mAb were used to immunoscreen hTg expression libraries. Only the same four mAb revealed immunoreactive clones corresponding to region 1149-1295 on the hTg primary sequence. After subcloning, this antigenic determinant was reduced to a 102-amino acid peptide (hTg region 1149-1250). The two different methodologies were coherent and complementary, and demonstrated that hTg sequence 1149-1250 is the target for this cluster of four mAb. Moreover, anti-hTg autoantibodies which cross-reacted with these mAb bound the 102-amino acid peptide. This epitope was the one most frequently detected by sera from autoimmune thyroid disease. The data confirm the presence of an immunodominant domain in the central part of the hTg molecule and suggest that this mAb epitope may be a powerful probe for the diagnosis of autoimmune thyroid disorders.


Characterization of the bovine peripheral lymph node homing receptor: a lectin cell adhesion molecule (LECAM).

The phenomenon of tissue-specific homing of lymphocyte populations has been most clearly shown in larger domestic animals, such as the sheep and cow, yet the molecular interactions which control these processes in these animals have not been defined. Here we tested the cross-reactivity of four anti-human peripheral lymph node homing receptor (LECAM-1) (also known as LAM-1, LEC-CAM-1, Leu-8, TQ-1, or human equivalent of gp90 MEL-14) antibodies on bovine lymphocytes. These antibodies stained all bovine neutrophils and monocytes, and variable numbers of peripheral blood lymphocytes, as determined by flow cytometry. In young calves (less than 1 month old) virtually all circulating lymphocytes expressed LECAM-1, whereas the percentage of positive lymphocytes in older animals (greater than 1 year) varied from 17%-67%. Bovine LECAM-1 was rapidly lost from the cell surface of PMA-activated and chymotrypsin-treated cells. Anti-LECAM-1 monoclonal antibody blocked greater than 80% of bovine lymphocyte binding to peripheral lymph node high endothelial venules (HEV). Since the lectin domain of LECAM-1 is thought to mediate lymphocyte-HEV adhesion, we sought to establish further the similarity of the bovine, mouse, and human molecules by comparing nucleotide sequences in this region of the molecule. The polymerase chain reaction (PCR) was used to specifically clone the bovine lectin domain from single-strand cDNA. Subsequent sequencing showed an identity of greater than 80% at the nucleotide level with the human and mouse molecules. The predicted amino acid sequences were also highly conserved. Though striking similarities were seen between the bovine, mouse and human molecule, indicating evolutionary conservation of this family of proteins, notable differences were detected. The nucleotide sequence of the bovine lectin domain predicts one additional N-linked glycosylation site compared to mouse and human. Preliminary analysis suggested a more tissue-restricted expression of LECAM-1 in the compared to the human and mouse, which correlates with a better separation of lymphocyte homing phenotypes seen in these larger animals. Virtually all peripheral lymph node lymphocytes in 6-month-old calves expressed LECAM-1, whereas, ileal Peyer's patch lymphocytes were predominantly negative. Finally, by testing anti-human LECAM-1 antibodies in a different species we have established the co-expression of antigenic epitopes on leucocyte LECAM-1 and a molecule(s) expressed by endothelial cells.


Mouse autoreactive gamma/delta T cells. I. Functional properties of autoreactive T cell hybridomas.

A minor population of dendritic epidermal T cells (DETC) express the V gamma 1.1C gamma 4V delta 6 T cell receptor and T cell clones and hybridomas derived from this subset constitutively secrete cytokines in culture secondary to recognition of an autoantigen. Activation of these autoreactive cells requires the use of the vitronection receptor (VNR) as an accessory molecule which interacts with the Arg-Gly-Asp-Ser (RGDS) sequence of extracellular matrix (ECM) proteins. We have compared the functional properties of C gamma 4+ hybridomas derived from newborn thymocytes and from adult spleen with the DETC hybridomas/lines in terms of their ability to secrete cytokines spontaneously and for the use of the VNR as an accessory molecule. Almost all the C gamma 4+ thymocyte hybridomas secreted cytokines spontaneously and in the majority of lines the most prominent cytokine secreted was granulocyte-monocyte colony-stimulating factor. In contrast, none of the four splenic C gamma 4+ hybridomas secreted cytokines spontaneously although all were capable of cytokine production following activation via the T cell receptor. Although the thymocyte hybridomas did not grow as adherent cell lines in culture, constitutive cytokine production required engagement of the VNR by its ligand in ECM proteins. In all cases, cytokine production could be inhibited by an anti-VNR monoclonal antibody as well as by soluble RGDS. The strong correlation of functional and molecular properties between thymocyte C gamma 4+ hybridomas and C gamma 4+ DETC suggests that the C gamma 4+ DETC may be of thymic origin and that cells with potential for autoreactivity residing in the thymus at birth may populate other peripheral locations in the mouse. The data also support the concept that the VNR, and possibly other integrins, play a role as accessory elements for autoreactive cells and may be essential for the regulation of such activity.


Inhibitory activity against sporozoites induced by antibodies directed against nonrepetitive regions of the circumsporozoite protein of Plasmodium vivax.

In previous studies, Saimiri sciureus boliviensis monkeys have been immunized with four recombinant proteins reproducing part of the circumsporozoite (CS) protein of Plasmodium vivax sporozoites (NS1(81) V20, rPvCS-1, rPvCS-2, rPv-CS-3), or with irradiated sporozoites of P. vivax Salvador I strain. To analyze the antibody response elicited against epitopes located outside the immunodominant repeat region of the CS protein, serum samples from these animals were tested for their ability to inhibit the in vitro development of liver stages of P. vivax VK247 strain, characterized from the other strains only by a specific repeat region on the CS protein. Results indicated that there is at least one protective B-cell epitope outside the repeat region of the CS protein of P. vivax sporozoites, and that this epitope can be expressed by irradiated sporozoites, rPvCS-1 and -3, but not by rPvCS-1 or NS1(81)V20. Therefore, we designed peptides from the amino acid sequences present both in rPvCS-2 and -3, but not included in the recombinant proteins rPvCS-1 and NS1(81)V20. Anti-peptide antibodies had no activity on the development of sporozoites of P. vivax Salvador I strain, into schizonts in primary culture of Saimiri monkey hepatocytes. In addition, antisporozoite antibodies did not react with any of the peptides. These results suggest that the in vitro inhibition observed in this study could depend upon the conformation of the CS protein. This study also demonstrates that antibody response to unnatural linear epitopes can be induced by immunization with recombinant proteins.


CD45 modulates T cell receptor/CD3-induced activation of human thymocytes via regulation of tyrosine phosphorylation.

Stimulation of thymocytes or mature T cells via the T cell receptor (TcR)/CD3 complex activates a cascade of processes inducing cells to enter the cell cycle. A key step is the activation of phosphatidylinositol-specific phospholipase C (PI-PLC) within seconds following TcR/CD3 stimulation, an event which is strongly enhanced by co-ligation of the CD4 (or CD8) accessory molecule with TcR/CD3. In contrast, co-ligation of CD45 inhibits the same TcR/CD3 responses. The machinery which couples the TcR/CD3 complex, CD4, and CD45 to PI-PLC appears to involve regulation of tyrosine phosphorylation, as the TcR/CD3 and CD4 receptors are associated with the tyrosine kinases p59fyn and p56lck, respectively, and CD45 has intrinsic tyrosine phosphatase activity. Here, we have examined the ability of CD45 to regulate signal transduction via TcR/CD3 in human thymocytes. Co-cross-linking CD45 to the TcR/CD3 complex strongly suppressed the tyrosine phosphorylation of several intracellular substrates normally seen following TcR/CD3 stimulation. This effect of CD45 was associated with inhibition of a rise in intracellular calcium following TcR/CD3 ligation. Since TcR/CD3 stimulation of mature T cells induces tyrosine phosphorylation of PLC gamma 1, we investigated this phenomenon in thymocytes, and asked whether ligation of CD45 might regulate this process. By immunoprecipitation we found that TcR/CD3 stimulation induced tyrosine phosphorylation of PLC gamma 1, an effect which was enhanced by co-cross-linking CD4 to TcR/CD3. In contrast, co-ligation of CD45 strongly blocked PLC gamma 1 phosphorylation induced by either stimulus. Consistent with previous findings in mature T cells, CD45 cross-linking was able to partially inhibit TcR/CD3-induced thymocyte proliferation when interleukin 2 was used as a second signal, but almost completely (80%-90%) blocked proliferation when anti-CD28 mAb was used as the second signal, suggesting that CD45 cross-linking may be able to block interleukin 2 production via the CD28 pathway. These effects of CD45 on TcR/CD3 signaling and proliferation in thymocytes point towards a potential role for this pathway in thymic selection.


T cell receptor diversity and activation markers in the V delta 1 subset of rheumatoid synovial fluid and peripheral blood T lymphocytes.

In the present study we have characterized the gamma/delta T cell receptor (TcR) population in synovial fluid (SF) and peripheral blood (PB) of patients with chronic inflammatory arthritis. By double staining we have shown that (a) synovial V delta 1+ cells have a high expression of activation markers CD45R0 ("memory cells") and HLA-DR as compared to PB, indicating a preactivated population of V delta 1-carrying T cells in vivo and (b) interleukin 2-induced expansion of synovial cells yields a high proportion of gamma/delta in most samples expressing predominantly the V delta 1 TcR. Junctional sequence analysis of the TcR delta chain from interleukin 2-expanded PB cell lines demonstrated a polyclonal V delta 1 population in three out of three samples. In SF cell lines three out of four samples were polyclonally expanded. In SF from one patient, however, a limited repertoire of expressed V delta 1 genes was found. Altogether, our data demonstrate the presence of preactivated V delta 1-expressing cells in the synovial compartment. This V delta 1 population is predominantly polyclonal, except in one patient where oligoclonally expanded V delta 1 cells were detected.


Characterization of rat encephalitogenic T cells bearing non-V beta 8 T cell receptors.

In this study, we demonstrate that T cell lines specific for a synthetic peptide representing sequence 87 to 99 of myelin basic protein (MBP) are encephalitogenic in Lewis rats. However, unlike syngeneic T cells specific for MBP residues 68 to 88 which exclusively use V beta 8 in their antigen receptors, these cells do not. None of the 10 T cell lines and T hybridomas specific for MBP (87-99) used V beta 8 in their T cell receptors. Our results document for the first time that rat encephalitogenic T cells do not exclusively use V beta 8 in T cell receptors that rat encephalitogenic T cells specific for MBP (87-99) are heterogeneous and that MBP (87-99) contains at least two epitopes for rat T cells.


Activation of natural killer cells via the Fc gamma RIII (CD16) requires initial tyrosine phosphorylation.

Triggering of the Fc gamma RIII (CD16) on natural killer (NK) cells by monoclonal antibodies or antibody-coated target cells stimulates a rapid phospholipase C (PLC)-mediated hydrolysis of inositol phospholipids and results in subsequent delivery of the lytic hit. The role of initial tyrosine phosphorylation in these events was investigated with a tyrosine protein kinase (TPK) inhibitor, genistein. At doses that inhibited CD16-triggered tyrosine phosphorylation of substrates in intact cells, genistein did not influence serine/threonine phosphorylation or target cell binding but prevented PLC activation, cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. These findings indicate that tyrosine phosphorylation is an early and critical event during receptor-mediated activation of the lytic machinery.


Intracellular Ca2+ release by flufenamic acid and other blockers of the non-selective cation channel.

We report in this paper using measurement of intracellular free Ca2+ with fura-2, that flufenamic acid and several related blockers of the 25 pS Ca(2+)-activated non-selective cation channel cause release of Ca2+ from an intracellular store other than the endoplasmic reticulum, possibly from mitochondria. A new compound, 4'-methyl-DPC, is found to be as effective in blocking non-selective cation channels as other flufenamate analogs but, like the parent compound, the non-selective cation channel blocker DPC, it does not cause release of Ca2+ from intracellular stores. DPC and 4'-methyl-DPC are thus the most suitable of the available blockers of non-selective cation channels for use in studies on the role of these channels in normal cell function.


Imidazole-SDS-Zn reverse staining of proteins in gels containing or not SDS and microsequence of individual unmodified electroblotted proteins.

A reverse staining procedure is described for the detection of proteins in acrylamide and agarose gels with and without SDS. Protein detection occurs a few minutes after electrophoresis. The sensitivity on acrylamide gels is higher than that of Coomassie blue staining either on acrylamide gels or on electrotransferred membranes. Sequencing of protein bands only detected by reverse staining on the gel and not by Coomassie blue is demonstrated.


Transcription mapping of the Ori L region reveals novel precursors of mature RNA species and antisense RNAs in rat mitochondrial genome.

We have identified new transcripts in the region surrounding the L-strand replication origin (Ori L) of rat liver mitochondrial DNA. In particular, we have detected previously unidentified intermediates of RNA processing on both the heavy and the light strands, such as precursors of the ND2 mRNA plus the Trp-tRNA and precursors of the tRNAs clustered in the Ori L region. This indicates that the mechanism of RNA processing in mitochondria proceeds step-wise producing a variety of precursors of the mature forms. The other striking finding is the detection of antisense RNA species in the region of L-strand replication. Since a variety of antisense transcripts were also found in the D-loop region of rat mitochondrial DNA, we suggest that they might play a regulatory role in the replication and expression of the mitochondrial genome.


A depolarization can trigger Ca2+ uptake and the acrosome reaction when preceded by a hyperpolarization in L. pictus sea urchin sperm.

The acrosome reaction (AR) is an exocytotic event that allows sperm to recognize and fuse with the egg. In the sea urchin sperm this reaction is triggered by the outer investment of the egg, the jelly, which induces ionic movements leading to increases in intracellular Ca2+ ([Ca2+]i) and intracellular pH (pHi), a K(+)-dependent transient hyperpolarization which may involve K+ channels, and a depolarization which depends on external Ca2+. The present paper explores the role of the hyperpolarization in the triggering of the acrosome reaction. The artificial hyperpolarization of Lytechinus pictus sperm with valinomycin in K(+)-free seawater raised the pHi, caused a small increase in 45Ca2+ uptake, and triggered some AR. When the cells were depolarized with KCl (30 mM) 40-60 sec after the induced hyperpolarization, the pHi decreased and there was a significant increase in 45Ca2+ uptake, [Ca2+]i, and the AR. This waiting time was necessary in order to allow the pHi change required for the AR to occur. Thus, the jelly-induced hyperpolarization may lead to the intracellular alkalinization required to trigger the AR, and, on its own or via pHi, may regulate Ca2+ transport systems involved in this process. Because of the key role played by K+ in the triggering of the AR, the presence and characteristics of ion channels in L. pictus isolated sperm plasma membranes are being explored. Planar lipid bilayers into which these membranes were incorporated by fusion displayed 85 pS single channel transitions which were cation selective.


An investigation of the specification of unequal cleavages in leech embryos.

Leech embryos develop via stereotyped cell divisions, many of which are unequal. The first division generates identifiable cells, blastomeres AB and CD, which normally follow distinct developmental pathways. When these two cells are dissociated and cultured in isolation, their fates remain distinct and are reminiscent of normal development, but their typical cleavage patterns are disrupted; cell AB undergoes relatively few cell divisions, giving rise to a variable number of macromeres and micromeres, while cell CD cleaves many times, usually forming a poorly organized set of macromeres, embryonic stem cells (teloblasts), and micromeres. We have investigated the hypothesis that the abnormal cleavage pattern of isolated CD blastomeres is due to removal of mechanical constraints normally imposed by cell AB. We find that when cell CD is constrained in vitro to mimic its in vivo shape, it cleaves more normally.


Mechanism of skin morphogenesis. I. Analyses with antibodies to adhesion molecules tenascin, N-CAM, and integrin.

To understand cell interactions during induction of skin appendages, we studied the roles of adhesion molecules N-CAM, tenascin, integrin, and fibronectin during feather development. Tenascin appeared in a periodic pattern on epithelia and was so far the earliest molecule detected in placodes. Three placode domains were identified: the anterior was positive for tenascin, the distal positive for N-CAM, and the posterior lacking both. Integrin appeared in dermal-epidermal junctions of placodes. In feather buds, sagittal sections revealed a transient anterior-posterior asymmetry with tenascin and N-CAM enriched in the anterior mesoderm. Tangential sections revealed a lateral-medial asymmetry with tenascin distributed in a ring shape and N-CAM in an "X" shape. Integrin was diffusely distributed within buds. Later tenascin and N-CAM were enriched in dermal papilla, the inducer of skin appendages. Perturbation of embryonic skin explant cultures with antibodies showed that anti-integrin beta 1 and anti-fibronectin blocked epithelial-mesenchymal interaction, anti-N-CAM caused uneven segregation of mesenchymal condensation, and anti-tenascin inhibited feather bud elongation. Dose-response curves showed gradual effects by these antibodies. The results indicated that these adhesion molecules are independently regulated and each contributes in different phases during morphogenesis of skin appendages.


Structural and developmental analysis of two linked myosin heavy chain genes.

We have identified at the molecular level two murine myosin heavy chain (MHC) genes which encode adult skeletal isoforms. As is the case for the murine cardiac MHC genes, the genes are closely linked, head to tail. To identify the isoform encoded by each gene, we located and sequenced the 3' termini and assessed the genes' temporal and tissue-specific expression patterns using probes specific for the 3' untranslated regions. These analyses indicate that the myosin encoded by the gene located 5' in the linked pair is the murine 2A isoform. The isoform encoded by the 3'-most gene of the linked pair appears to encode an additional isoform that is expressed in a number of adult skeletal muscles. The pattern of expression of the 3'-most gene indicates that it is tightly controlled developmentally. Transcripts from this gene, when compared to those from the MHC2A and beta-MHC genes, are the predominant MHC transcripts found in the diaphragm, tongue, soleus, and masseter, indicating that it encodes a major skeletal MHC isoform.


Effects of imidazole derivatives on cytochromes P450 from human hepatocytes in primary culture.

The expression of several forms of cytochrome P450 including P450 1A2, 2D6, 2E1, and 3A was investigated in human hepatocytes maintained in primary culture for 96 h in the absence or presence of 50 microM of various imidazole derivatives. These included ketoconazole, clotrimazole, miconazole, fluconazole, secnidazole and metronidazole. In addition, the typical inducers rifampicin and beta-naphthoflavone were used for comparison. Western and Northern blot analysis of microsomes and RNA prepared from these cultures as well as de novo synthesis experiments revealed that, among the imidazole derivatives tested, only clotrimazole was a strong rifampicin-like inducer of P450 3A. The expression of the other forms of P450 tested was not affected by the treatments. Analysis of the inhibition of 13 monoxygenase activities, including ethoxyresorufin and phenacetin O-deethylases, coumarin 7 alpha-, lauric acid 11- and 12-, mephenytoin 4-, debrisoquin 4-, and aniline hydroxylases, benzphetamine, aminopyrine, mephenytoin and erythromycin demethylases, and cyclosporin oxidase (representative of 10 different forms of P450 in human liver microsomes) revealed that ketoconazole was a strong and selective in vitro inhibitor of P450 3A (cyclosporin oxidase) with a Ki less than 1 microM. Clotrimazole and miconazole were also strong inhibitors of P450 3A-mediated activities in contrast to the other imidazole derivatives.


Ruthenium red delays the onset of cell death during oxidative stress of rat hepatocytes.

Our objective was to determine if ruthenium red protects against lethal oxidative injury of rat hepatocytes. tert-Butyl hydroperoxide, 100 mumol/L, was used to produce oxidative stress. After 2 hours of oxidative stress, cell viability was greater with than without 25 mumol/L ruthenium red (37% vs. 4.6%; P less than 0.01). Despite this cytoprotection, ruthenium red did not alter the rate or extent of glutathione depletion, malondialdehyde generation, or adenosine triphosphate depletion. In contrast, ruthenium red did retard loss of the mitochondrial membrane potential (78% vs. 42% within 30 minutes; P less than 0.01). However, the protective effect of ruthenium red could not solely be explained by preserving the mitochondrial membrane potential. Indeed, ruthenium red still improved cell survival after 2 hours of exposure to 10 mumol/L carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler (39% vs. 13%; P less than 0.01). Cytosolic free calcium values did not change during the uncoupling of mitochondria, suggesting that the cytoprotective properties of ruthenium red cannot be explained by blocking mitochondrial calcium transport. Ruthenium red did inhibit proteolysis after 2 hours of exposure to tert-butyl hydroperoxide (434 +/- 62 vs. 242 +/- 20 nmol/10(6) cells; P = 0.016) or CCCP (236 +/- 50 vs. 99 +/- 38 nmol/10(6) cells; P = 0.04). The results indicate that ruthenium red appears to protect against hepatocellular injury by inhibiting degradative proteolytic activity. It is concluded that proteolysis may be an important mechanism contributing to lethal oxidative injury of hepatocytes.


Nucleotide sequences of 5-1-1 of hepatitis C virus in patients with chronic liver disease.

The discovery of hepatitis C virus (HCV) has led to the development of an assay against a viral peptide (C100-3), which is now used worldwide. It has been shown that the majority of HCV-infected individuals are positive for the antibody. However, there are patients who are repeatedly seronegative for the antibody despite the presence of HCV RNA in the serum. The nucleotide sequences of 5-1-1 (a major epitope of C100-3) obtained from five antibody-positive patients and five negative patients with chronic liver disease were studied. The nucleotide identities of the seropositive and seronegative patients with HCV prototype sequence were 80.6% and 81.8%, respectively, not showing much difference in the nucleotide sequence of the 5-1-1 region. Moreover, no marked differences were noted in the deduced amino acid residues and the hydropathy profiles between the two groups. These data suggest that absence of the antibody in HCV carriers are not due to variations of major epitopes but are probably due to immunological incompetence against the synthetic C100-3 peptide.


The ontogeny and distribution of neuropeptides in the human fetal and infant esophagus.

The innervation and neuropeptide expression of fetal and infant human esophagus were studied. Esophageal samples (n = 30) from 8 weeks' gestation to 28 months of age were immunostained using antisera to general and specific neuronal antigens, and the results were quantified using computer-assisted image analysis. Nerve protein (protein gene peptide 9.5 and synaptophysin) and glial cell protein (S100) immunoreactivities were present by 8 weeks' gestation in primitive cell bodies and fibers in the outer layers of the esophagus. Immunoreactivity for peptides was first detected in fibers at 11 weeks' gestation in myenteric plexus and at 13 weeks' gestation in muscle. Peptide-immunoreactive cell bodies were not seen until 13-15 weeks. A pattern of immunoreactivity for neuropeptides comparable with that seen in mature neonates and infants was present by 22 weeks of gestational age. The percentage area of protein gene peptide 9.5-immunoreactive and vasoactive intestinal peptide-immunoreactive nerve fibers increased from low levels to 3.68% and 0.27%, respectively, at 13 weeks and peaked at 18 weeks (10.50% and 4.74%). These findings provide a foundation for future research into the contribution of neuropeptides to pediatric esophageal dysmotility.


Hepatitis B virus envelope epitopes: gene assembly and expression in Escherichia coli of an immunologically reactive novel multiple-epitope polypeptide 1 (MEP-1).

A novel synthetic 323-bp gene with the open reading frame of a multiple-epitope polypeptide has been assembled and cloned. The gene is engineered by contiguous alignment of selected epitopes and functional domains of the hepatitis B virus envelope proteins separated by pairs of glycine residues. High-level bacterial production of this 100-amino acid (approx. 10 kDa) protein has been achieved and the gene product is stable. ELISA and Western blot experiments using epitope-specific antisera confirm that the corresponding epitopes are present in the engineered protein.


Isolation and characterization of a human gene that encodes a new subclass of protein tyrosine kinases.

We describe the isolation and cDNA sequence of a novel human gene, which is distinct from all known members of the human src family of proto-oncogenes. In contrast to these, an autophosphorylation site corresponding to Tyr416, as well as the equivalent of Tyr527 in p60c-src, are missing in the amino acid (aa) sequence deduced from this gene. Furthermore, no N-terminal myristylation site is found. Our human clone is 98% identical at the aa level to a gene which was isolated independently from neonatal rat brain and was termed csk for c-src kinase. We, therefore, propose to designate the present human gene CSK. In Northern blot experiments, CSK was found to be expressed in human lung and macrophages. Due to its extreme conservation across species barriers, the CSK product is likely to exert important regulatory functions. On the basis of its expression in tissues, not typically expressing high c-src levels, it can be assumed that its regulatory role is more general and may also involve other tyrosine kinases.


Prevention of post-ischemic brain lipid conjugated diene production and neurological injury by hydroxyethyl starch-conjugated deferoxamine.

Hydroxyethyl starch conjugated deferoxamine (DFO) was administered to rats following resuscitation from 6.5 min cardiac arrest (CA) in an attempt to prevent the iron-catalyzed production of oxygen free radicals which may lead to neurologic injury and ultimately death following restoration of spontaneous circulation (ROSC). Brain conjugated dienes were analyzed spectrophotometrically 4 and 24 hr following ROSC, and were found to be significantly elevated when compared to non-ischemic controls. Hydroxyethyl starch-DFO treated rats demonstrated no increased conjugated diene production at either period. Neurologic injury was significantly less in drug treated rats surviving 24 or 72 hours when compared to controls. While mortality was similar in drug treated or control rats for the first 24 hours following ROSC, delayed mortality (days 1-10) was significantly less in drug treated animals, presumably as a result of neurologic protection afforded by post-ischemic drug administration. Administration of DFO conjugated to hydroxyethyl starch appears to modulate the neurologic injury which occurs during brain ischemia and reperfusion.


Effects of FK506 and cyclosporin A on cytokine production studied in vitro at a single-cell level.

Mononuclear cells obtained from human blood were mitogen or antigen activated in vitro in the presence or absence of FK506 or cyclosporin A (CsA). Cytokine production was studied at a single-cell level by ultraviolet (UV) microscopy of fixed permeabilized cells using cytokine-specific monoclonal antibodies (mAb). Phenotypic characterization of the monokine-producing cells was achieved by two-colour immunofluorescent staining. Cytokine production after antigen activation with Staphylococcus aureus enterotoxin A (SEA) was significantly reduced. FK506 or CsA inhibited SEA-induced tumour necrosis factor-alpha (TNF-alpha) production both in monocytes (P less than 0.01) and in lymphocytes (P less than 0.001), at a drug concentration of 1-25 ng/ml for FK506 and 100-500 ng/ml for CsA. Lymphocyte synthesis of interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and TNF-beta after SEA activation was also significantly reduced by either of the drugs. In contrast, endotoxin-induced monokine production (TNF-alpha and IL-6) after lipopolysaccharide (LPS) stimulation was unaffected by FK506 or CsA even when added in concentrations as high as 1000 ng/ml. When the cells were stimulated by phorbol ester (phorbol 12-myristate 13-acetate, PMA) plus calcium ionophore (ionomycin), FK506 and CsA inhibited, in a dose-dependent manner, the production of IL-2, IL-4, IL-5, IFN-gamma and TNF-alpha. The 50% inhibitory concentration (IC50) for FK506 or CsA on the cellular synthesis of the various cytokines varied between 0.6 and 1.0 ng/ml and 20 and 60 ng/ml, respectively. Further stimulation by addition of anti-CD28 mAb to the cultures resulted in an augmented IL-2 and IFN-gamma production which was resistant to both FK506 and CsA. This report delineates extensive similarities between the two drugs in mechanisms of immunosuppression by blockade of identical interleukin production. Depending on the mode of cell activation the two drugs inhibited not only cytokine production in lymphocytes but also antigen-induced monokine (TNF-alpha) production in macrophages, although the optimal immunomodulatory effect of FK506 was achieved at a concentration approximately 50-fold lower than that of CsA.


Anti-platelet autoantibodies from ITP patients recognize an epitope in GPIIb/IIIa deduced by complementary hydropathy.

Idiopathic thrombocytopenic purpura (ITP) is a frequent platelet disorder due to the presence of anti-platelet autoantibodies. Recently a fibronectin/fibrinogen receptor in platelets, integrin GPIIb/IIIa, has been implicated as the antigen in chronic ITP. To examine the epitopes involved in the autoimmune response against GPIIb/IIIa we have used concepts from the complementary hydropathy principle. We used the peptide Trp-Thr-Val-Pro-Thr-Ala, WTVPTA (deduced from the complementary nucleotide sequence to that which codes for the Arg-Gly-Asp, RGD, domain in fibronectin), to test the immunologic activity of ITP sera. Sera from 31 patients with clinically defined ITP were tested in ELISA for reactivity towards WTVPTA and affinity purified GPIIb/IIIa. Seventeen sera (57%) reacted strongly with the glycoprotein complex, five of which reacted with the peptide. By affinity chromatography of one of these sera, we were able to show that antibodies that bind to the peptide are within the population that binds to GPIIb/IIIa. Liquid phase competition experiments revealed that binding of ITP serum to WTVPTA was inhibited only by a hydropathically compatible peptide. Our data indicate that autoantibodies can bind to hydropathically generated antigenic determinants and thus, render these peptides clinically important as diagnostic tools.


Phenotypic analysis of complement receptor 2+ T lymphocytes: reduced expression on CD4+ cells in HIV-infected persons.

While expression of complement receptor 2 (CR2) (CD21) on some CD4+ cell lines renders them more susceptible to infection by complement-treated human immunodeficiency virus (HIV), coexpression of CR2 and CD4 on peripheral blood lymphocytes has not, until recently, been observed. Several recent studies, however, have found that human T lymphocytes express low levels of CR2. Additionally, complement treatment of HIV before addition to these cells has been reported to increase virus expression in peripheral blood lymphocyte cultures. These findings suggest that complement-mediated enhancement of infection of human T cells could occur in vivo and have prompted us to examine both the phenotypic properties of CD4+CR2+ T cells in healthy persons and the expression of CR2 on CD4+ lymphocytes during HIV infection. As was previously reported, we observed CR2 on a proportion (10-50%) of both CD8+ and CD4+ T cells. Approximately half of CD4+CR2+ cells expressed the memory cell markers CD45RO and CD29, 80% expressed the naive marker CD45RA, while 22% expressed CD25. These values were not substantially different from total CD4+ cells. Stimulation of lymphocytes with phytohaemagglutinin (PHA), OKT3 or calcium ionophore but not with phorbol myristate acetate (PMA) or interleukin-2 (IL-2) decreased expression of CR2 on CD4 cells by half over a 3-day culture period. The per cent of CD4+ cells expressing CR2 was significantly decreased in patients with asymptomatic and symptomatic HIV infection compared to uninfected control donors (P = 0.0001). In contrast, the decrease in CR2 expression was not observed with CD8+ lymphocytes from HIV-infected persons. These results confirm that CR2 is expressed on human T lymphocytes and suggest that a subset of CD4+ lymphocytes is selectively affected in HIV-infected individuals.


Antigen-specific and polyclonal CD4+ lamina propria T-cell lines: phenotypic and functional characterization.

Surface phenotype and function of lamina propria CD4+ T cells have been evaluated. In addition, long-term, antigen-specific and polyclonal lamina propria CD4+ T-cell lines have been generated and characterized. Lamina propria CD4+ T cells represent approximately 30% of lamina propria lymphocytes and are responsive to a variety of T-cell mitogens, including anti-CD3, concanavalin A, phytohaemagglutinin and pokeweed mitogen. In each case, however, lamina propria T cells are less responsive to these mitogens than spleen T cells. Freshly isolated lamina propria T cells produce substantial amounts of interleukin-2 (IL-2), interleukin-4 (IL-4), gamma interferon and to a lesser extent interleukin-5 (IL-5). Antigen-specific lamina propria CD4+ T-cell lines were generated by orally immunizing animals with antigen (KLH) in conjunction with cholera toxin as an oral adjuvant. Polyclonal lamina propria CD4+ T-cell lines were generated from unimmunized animals using anti-CD3 as a polyclonal stimulus. Both antigen-specific and polyclonal CD4+ T-cell lines were Thy-1+, alpha beta TCR+ and CD8-. The antigen-specific CD4+ T-cell line when stimulated by anti-CD3 and PMA produces predominantly IL-2, IL-4 and gamma interferon, with very little IL-5. In contrast, the polyclonal CD4+ T-cell line when similarly stimulated produces predominantly IL-4 and IL-5, with very little IL-2 and no detectable gamma interferon. In summary, lamina propria CD4+ T cells have been evaluated and in vitro conditions have been determined for successful generation of lamina propria CD4+ T-cell lines.


T-helper functions in lines of mice selected for high or low antibody production (Selection III): modulation by anti-CD4+ monoclonal antibody.

T-helper function was evaluated in mice genetically selected for high (H) or low (L) antibody (Ab) responsiveness to Salmonella flagellar antigen (Ag) (Selection III). In this Selection as opposed to what was demonstrated in Selections I, II and IVA, the interline difference was not proven to be based upon the modification of Ag processing and presentation at macrophage level. CD4+/CD8+ lymph node ratio is similar in HIII and LIII mice, both lines being equally susceptible to in vivo depletion of CD4+ T cells by GK 1-5 monoclonal antibody (mAb) treatment. Nevertheless, the Ab responsiveness of the two lines was differently modulated by GK 1-5 mAb: the inhibition of Ab responses to various Ag required lower mAb doses and was long lasting in LIII as compared to the transient effect of higher mAb doses observed in HIII. LIII mice were also refractory to Salmonella-induced reversion of GK 1-5 mAb inhibition. Moreover, in vitro specific I proliferation was constantly lower in LIII, though its IL-2 production was unexpectedly similar to that of HIII T cells. Results of in vivo and in vitro experiments are thus consistent with a defective response of T-helper cells to immunogenic challenge in LIII mice.


Primary and secondary human in vitro T-cell responses to soluble antigens are mediated by subsets bearing different CD45 isoforms.

A culture system has been developed which consistently supports in vitro proliferative responses to conventional soluble antigens by human CD4+ T cells from non-immunized donors. T cells exposed to an antigen in primary cultures could be restimulated in vitro in an antigen-specific manner to give secondary responses with greater magnitudes and a more rapid onset than the initial reaction. To characterize further the responding T-cell population in primary compared with secondary reactions, T cells were depleted of CD45RA+ or CD45RO+ cells and stimulated with recall and non-recall antigens. It was found that the soluble non-recall antigen keyhole limpet haemocyanin did not stimulate CD45RO+ T cells, yet induced strong proliferative responses from CD45RA+ T cells. Conversely, it was confirmed that human CD45RO+ T cells respond to the recall antigen-purified protein derivative from Mycobacterium tuberculosis. Cell mixing experiments indicated that CD45RO+ T cells are unlikely to have any suppressive effect on the reactivity of CD45RA+ cells to non-recall antigens. These data provide new support for the hypothesis that CD45RA+ represents the naive and CD45RO+ the memory phenotype of human CD4+ T cells.


Pattern of lectin binding to murine T lymphocytes.

Lectins can be used to detect changes in cell-surface glycosylation. In this paper we examine the lectin-binding characteristics of murine T cells as measured by flow cytometry. A large panel of labelled lectins was used to stain naive, activated and resting murine T cells. Some lectins did not detectably bind any T cells, some bound to all T cells and some bound to only a subset of splenic T cells. Three lectins which preferentially bind to previously activated T cells were identified: Bandeiraea simplicifolia BS-I, Bauhinia purpurea and Lycopersicon esculentum. In addition, two lectins were found to bind preferentially to activated T cells: Glycine max and Triticum vulgaris. Finally, no differences were found in the ability of a large panel of lectins to bind to Th1 and Th2 clones. Lectin binding may be a powerful tool for distinguishing naive, activated and memory T cells.


A Tcra congenic mouse: V alpha epitope expression is influenced by both Tcra haplotypes and background genes.

A T-cell receptor alpha chain locus (Tcra) congenic mouse is described. The Tcraa haplotype of BALB/c (donor strain) was bred on to B10.D2 (background strain, Tcrab haplotype) by using a Bgl I Tcra-C restriction fragment length polymorphism. Tcraa/b heterozygous offspring from the eleventh backcross generation were brother-sister mated to obtain Tcra-Ca homozygous animals. The resulting congenic line, B10.D2.C-Tcraa/Bo carries a recombination between the Tcra and the hr loci; thus, the transferred differential segment is the centromeric 18-27 cM of the BALB/c chromosome 14. Analysis with a multitude of Tcra-V and Tcrd-V probes demonstrates that the complete Tcraa haplotype is contained within this differential segment. Lymph node T cells of BALB/c (Tcraa) B10.D2 (Tcrab) and B10.D2.C-Tcraa were stained with anti-V alpha 8 (KT50, KT65), anti-V alpha 3.2 (RR3-16) and anti-V alpha 11.1 and 2 (RR8-1) monoclonal antibodies. We find that the frequencies of V alpha epitope expression are highly Tcra haplotype-dependent even though an influence of background genes is also observed. Thus, Tcra-V germline differences may possibly influence the T cell repertoire, in addition to the already well known positive and negative thymic selections. Tcra haplotype does not influence the frequencies of V beta utilization. However, BALB/c mice have fewer V beta 11+ T cells than B10.D2 and B10.D2-Tcraa, therefore, the BALB/c genome must harbor a V beta 11 deleting gene(s) in addition to those described so far.


Chromosome 14 in B10.A(18R) mice is recombinant and includes Tcra-Va alleles.

Analysis of mouse Tcr genes has previously defined at least five different Tcra-V haplotypes among inbred strains of mice. For mice of the Tcra-Vb haplotype, including C57BL/10 (B10), T-cell expression of the Tcra-V11 gene subfamily can be detected with a monoclonal antibody, 1.F2. In the course of further characterizing the specificity of 1.F2, we found that it fails to recognize Tcra-V11-expressing T-cell hybrids derived from the B10 congenic strain, B10.A(18R)/SgIcr. Moreover, staining analysis indicated that the Va11 epitope recognized by 1.F2 is not expressed by peripheral T cells from several different B10.A(18R) colonies with the exception of that at the Research Institute of Scripps Clinic. Nucleotide sequences were determined for cDNA representing rearranged Tcra-V11 genes from two independent, B10.A(18R)/SgIcr derived T-cell hybrids. The two Tcra-V11 gene segments were identical and the predicted amino acid sequence differed by at least five residues from Tcra-V11 sequences previously obtained from B10.A mice. Southern blot analysis of restriction fragment length polymorphisms (RFLP) associated with Tcra-V11, as well as Tcra-V1, subfamily genes revealed that the B10.A(18R) mouse has inherited Tcra-Va alleles rather than the expected Tcra-Vb alleles from the B10 strain. RFLP analysis of the Rib-1 locus, located in close proximity to the Tcra locus on chromosome 14, showed that B10.A(18R) carries the Rib-1b allele from B10. These results indicate that the B10.A(18R) mouse has inherited a recombinant chromosome 14 with a recombination event having occurred between the Rib-1 locus and the Tcra-V gene subfamilies examined. Inheritance of Tcra-Va alleles in B10.A(18R) probably originated from strain 129/J which breeding records show was used in the first cross with B10.A in the production of B10.A(18R) and which we found exhibits Tcra-V11a RFLPs.


Distribution of individual components of basement membrane in human colon polyps and adenocarcinomas as revealed by monoclonal antibodies.

Double-label immunofluorescence was used to monitor basement-membrane composition and integrity in 22 human colon polyps, 36 adenocarcinomas and 2 metastases. Cryostat sections were stained with polyclonal anti-laminin anti-serum combined with monoclonal antibodies (MAbs) to all major basement-membrane components (laminin, entactin/nidogen, collagen type IV and large heparan sulfate proteoglycan), as well as to keratin 8. In all adenocarcinomas, including mucinous, basement membranes were altered more at the invasive front than in the parenchyma. The degree of this alteration was inversely correlated with the level of tumor differentiation. An uncoordinated loss of basement membrane components (dissociation of markers), previously described by us in rat colon adenocarcinomas, was also found in human tumors. In the great majority of adenocarcinomas a pronounced stromal reaction was seen. It was manifested by the presence of fibrillar deposits of basement-membrane components, mainly of collagen type IV and/or heparan sulfate proteoglycan. This reaction was never observed in polyps and may be derived from myofibroblasts reported to accumulate in colon cancer stroma. The combined use of antibodies to basement-membrane components and to a specific keratin may constitute an adequate immunohistochemical test for the presence of invasion, and may be useful in the histologic analysis of polyps, especially in dubious cases.


Tissue-specific regulation of inflammation.

Proteins of the complement system are important effectors and modulators of inflammation. The complement cascade is triggered by microbes, tissue debris, and specific antibodies. Serum complement proteins are derived primarily from liver, but extrahepatic complement synthesis is important in homeostasis and in local host defenses. Tissue-specific regulation of expression of complement genes is governed by mechanisms similar to those that regulate other "acute phase reactants." That is, tissue injury or infection elicit changes in expression of these acute phase proteins, which, although variable in kinetics, magnitude, and direction, are a consequence of an elaborate system of cell-to-cell communication. This communication is mediated via a complex network of cytokines, including the interferons, interleukins, several growth factors, and sex hormones. The cell biological and molecular biological details of these mechanisms are now under active investigation. An understanding in molecular terms of the balance between proinflammatory and counterregulatory forces on complement gene expression should provide new insight into the functions of complement and the design of novel therapies for disorders of inflammation.


Increased negativity of interstitial fluid pressure in rat trachea in dextran anaphylaxis.

This study shows that, in rat trachea, dextran anaphylaxis is associated with increased negativity of interstitial fluid pressure (Pif) as measured with sharpened glass capillaries (tip diameter 3-7 microns) connected to a servo-controlled counterpressure system. Experiments were carried out in pentobarbital-anesthetized Wistar-Møller rats. Pif in the control situation was -2.5 +/- 0.38 (SD) mmHg. The mean pressure in animals killed 2 min after initiation of the anaphylactic reaction by injection of 1 ml of 10% Dextran 70 in 0.9% NaCl was -10.3 +/- 2.6 mmHg. In another experimental series, interstitial fluid volume was measured after dextran administration but without inducing circulatory arrest. Interstitial fluid volume increased from 0.94 +/- 0.16 to 1.56 +/- 0.42 ml/g dry wt after 10 min to 1.57 +/- 0.30 and 1.10 +/- 0.27 ml/g dry wt after 30 and 60 min, respectively. The increased negativity in Pif in tracheal mucosa in the early phase of dextran anaphylaxis will markedly increase the transcapillary net filtration pressure in the initial phase of edema development.


Growth of human renal cortical tissue on collagen gel.

A model system for 3-dimensional "native-state" culture of tissues on collagen gels (Proc. Natl. Acad. Sci. USA 86:2013-2017; 1989) has been applied in this study to histologically normal human renal cortical tissue from 11 patients undergoing nephrectomy for renal cell carcinoma elsewhere in the kidney. Microbial contamination occurred in 12/90 cultures, the rest (78) were studied by visual inspection, histology, immunohistochemical analysis for pankeratin (epithelial cell origin), vimentin (mesenchymal cell origin), and p-glycoprotein (associated with proximal tubules), transmission electron microscopy (EM), incorporation of tritiated thymidine (3HTdR). In the first 10 days, explants showed 3HTdR-labeled cells in tubule structures. The surrounding gel was invaded by cells forming tubule structures, sometimes with basement membrane. Some of these cells showed labeling by 3HTdR and immunostaining positive for pankeratin and p-glycoprotein. EM showed well-polarized epithelial cells in tubule structures with tight junctions, interdigitating lateral processes, and microvilli characteristic of proximal and distal convoluted tubules. 3HTdR-labeled cells in tubule structures were observed even 2 mo. after Passage 1, 6 mo. after the initial explantation. Tubule growth was most active and fibroblast proliferation was negligible from 2 to 4 wk postexplantation. The proliferation of tubulelike cells and formation of tubulelike structures in this system represents an opportunity to study human renal cortical tissue in vitro, under conditions more closely resembling in vivo circumstances than are present in other in vitro systems suitable for long-term study. This model has potential use for in vitro toxicology studies and studies of renal physiology.


SK HEP-1: a human cell line of endothelial origin.

SK-HEP-1 is an immortal, human cell line derived from the ascitic fluid of a patient with adenocarcinoma of the liver. We have determined that these cells are of endothelial origin. Despite the location of the tumor from which SK HEP-1 was derived, the cell line does not have properties of hepatocytes. Northern blot analysis of total cellular RNA shows no messenger RNA for the hepatic-specific proteins albumin, alpha-fibrinogen, or gamma-fibrinogen. Endothelial characteristics are seen by transmission electron microscopy. These features include numerous pinocytotic vesicles, electron dense granules consistent with Weibel-Palade bodies, and abundant intermediate filaments, identified immunocytochemically as vimentin. Cultures grown on plastic dishes grow in bundles of polygonal to spindle-shaped cells. Proteins characteristic for endothelial cells are identified by immunocytochemistry. Addition of basement membrane material (Matrigel) or type I collagen to the cultures induces these cells to organize into a tubular network.


Growth of sebaceous cells in monolayer culture.

Rat preputial cells were grown in an epithelial cell primary monolayer culture system identical to that used for culturing epidermal cells, which were studied for comparison. Despite similar appearance when observed by phase contrast microscopy, other features identified the preputial cells as a unique epithelial cell population. Preputial cells grew as a relatively small number of large colonies, formed domes before confluence, and expressed a specific acinar keratin, K4, which had previously been found in human sebaceous glands. In addition, preputial cells formed fewer cornified envelopes than epidermal cells, too few to discern the reduction of envelope formation by retinoic acid treatment in vitro which was found in epidermal cells. Rat preputial cells in monolayer culture, therefore, are a promising model for studying the effects of hormones on sebaceous cell growth and differentiation.


Isoleucyl-tRNA synthetase from the ciliated protozoan Tetrahymena thermophila. DNA sequence, gene regulation, and leucine zipper motifs.

We have determined the nucleotide sequence of a protozoan aminoacyl-tRNA synthetase. The isoleucyl-tRNA synthetase (ileRS) gene [ilsA; formerly cupC, Martindale, D. W., Martindale, H. M., and Bruns, P. J. (1986) Nucleic Acids Res. 14, 1341-1354] from the ciliate Tetrahymena thermophila was sequenced and found to have eight introns, four transcription start sites, and a putative polypeptide of 1081 amino acids. A polypeptide 20 amino acids longer could be made if a transcribed in-frame ATG close to the start sites and with suboptimal sequence context is used. This gene was identified through hybridization and amino acid sequence similarity to the previously cloned and sequenced ileRS (cytoplasmic) gene from Saccharomyces cerevisiae [Englisch, U., Englisch, S., Markmeyer, P., Schischkoff, J., Sternbach, H., Kratzin, H., and Cramer, F. (1987) Biol. Chem. Hoppe-Seyler 368, 971-979; Martindale, D. W., Gu, Z. M., and Csank, C. (1989) Curr. Genet. 15, 99-106] with which it shares 47% of its amino acids. We also compared it to ileRS genes from E. coli and an archaebacterium. Two leucine zippers motifs were identified in the carboxyl-terminal domain of the polypeptide; one of these motifs is in the same area as the zinc finger motif found in the E. coli enzyme. The transcription pattern of the ilsA gene was monitored under various culture conditions and parallels changes in protein synthesis.


Gene expression during 3T3-L1 adipocyte differentiation. Characterization of initial responses to the inducing agents and changes during commitment to differentiation.

The mouse 3T3-L1 fibroblastic cell line rapidly differentiates to an adipocyte phenotype when post-confluent cells are treated for 48 h in fetal calf serum-containing medium supplemented with 1 microM dexamethasone (D), 0.5 mM methylisobutylxanthine (M) and 10 micrograms/ml insulin (I). D and I act synergistically to commit the cells to differentiate 24-48 h after initiating treatment, and this is blocked by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. In order to identify cellular proteins involved in the differentiation process we analyzed differentiating 3T3-L1 cells using two-dimensional electrophoresis on large format gels. We observed changes in over 300 proteins during differentiation (over 100 within 5 h of initiating differentiation) and many of these are also changed at the level of mRNA (by analysis of in vitro translation products). About 75% of the initial changes were maximally induced by treatment with a combination of M and I, while no more than 10 proteins and their corresponding mRNAs were maximally induced by D within 3.5 h. Another 10 proteins were synergistically regulated by the combination of all three agents (DMI) within 3.5 h. Additional species were induced at later times. Five of these were synergistically induced by treatments that lead to differentiation, were first expressed at elevated levels during commitment and remained elevated in fully differentiated adipocytes. One or more of these proteins could well have a functional role in the commitment to and/or expression of the adipocyte differentiation program.


Biochemical and molecular genetic analyses on placental aromatase (P-450AROM) deficiency.

Biochemical and molecular genetic studies were made on a case of placental aromatase (P-450AROM) deficiency. Of the enzymes participating in the electron transport system of placental microsomes, only aromatase activity was decreased specifically in the patient, being less than 0.3% of the normal activity. Northern and Western blotting analyses showed that the transcription of the aromatase gene and the translation of its mRNA proceeded normally in the placenta of this patient. However, aromatase cDNA isolated from a placental cDNA library of the patient was found to have an insert of 87 base pairs, encoding 29 amino acids in frame with no termination codon. The insert was located at the splicing point between exon 6 and intron 6 of the normal aromatase gene, and the extra DNA fragment was the first part of intron 6, except that its initial GT was altered to GC. These findings indicated that in this patient with aromatase deficiency, splicing between exon 6 and intron 6 did not occur at the normal position because of a point mutation in its consensus sequence and was forwarded to GT in the next cryptic consensus sequence 87 base pairs downstream according to the canonical GT/AG rule, resulting in translation of an abnormal protein molecule with 29 extra amino acids. During the transient expression in COS-7 cells, the aromatase cDNA of the patient was found to produce a protein with a trace of activity. This is the first report of a genetic defect for aromatase deficiency.


Biochemical properties of chimeric skeletal and smooth muscle myosin light chain kinases.

The molecular and biochemical properties of myosin light chain kinases from chicken skeletal and smooth muscle were investigated by recombinant DNA techniques. Deletion of the amino-terminal region of either the smooth or skeletal muscle myosin light chain kinase resulted in a decrease in Vmax with no significant change in Km values for light chain substrates. Skeletal/smooth muscle chimeric kinases were inactive when a 65-residue region amino-terminal of the catalytic core was exchanged between the two forms. Changing alanine 494 to glutamic acid within this region in the chicken skeletal muscle myosin light chain kinase increased the Km values for light chains 10-fold. These results are consistent with the hypothesis that the region amino-terminal of the catalytic core in myosin light chain kinases is involved in light chain recognition. A skeletal muscle kinase which contained the smooth muscle calmodulin binding domain remained regulated by Ca2+/calmodulin. Thus, the calmodulin binding domains of smooth and skeletal muscle myosin light chain kinases share structural elements necessary for regulation.


Expression, synthesis, and phosphorylation of HSP28 family during development and decay of thermotolerance in CHO plateau-phase cells.

We investigated a correlation between development of thermotolerance and expression, synthesis, or phosphorylation of HSP28 family in CHO plateau phase cells. After heating at 45.5 degrees C for 10 min, thermotolerance developed rapidly and reached its maximum 12-18 hr after heat shock. This acquired thermal resistance was maintained for 72 hr and then gradually decayed. In parallel, the levels of three 28 kDa heat shock proteins, HSP28a along with its two phosphorylated isoforms (HSP28b,c), increased and reached their maximum 24-48 hr after heat shock. The levels of these polypeptides, except HSP28c, remained elevated for 72 hr and then decreased. The level of HSP28 mRNA increased rapidly and reached its maximum 12 hr after heat shock. However, unlike thermotolerance and the levels of HSP28 family proteins, the level of HSP28 mRNA decreased rapidly within 72 hr. These results demonstrate a correlation between the amount of intracellular HSP28 family proteins and development and decay of thermotolerance.


Alpha-fetoprotein-mediated uptake of fatty acids by human T lymphocytes.

The binding to resting and activated T lymphocytes of two radiolabelled fatty acids (oleic and arachidonic) was studied in the presence or in the absence of alpha-fetoprotein (AFP) as carrier protein. Fatty acid binding by resting and activated T lymphocytes was determined at 4 degrees C as a function of the concentration of fatty acid and AFP. Under the conditions employed, the following observations were made: (1) in the presence of AFP, fatty acids (oleic and arachidonic acid) are bound to cells by a two-component pathway; one is a saturable process, evidenced when the fatty acid to AFP (FA/AFP) molar ratio was fixed at 1 and the concentration of the fatty acid and the protein varied from 0.1 to 3.2 microM, and the second is a nonsaturable function of FA/AFP molar ratio and was linearly related to the unbound fatty acid concentration in the medium over the entire range studied; (2) in the absence of AFP, the nonsaturable process appears to be the only component of fatty acid binding; 3) at all tested concentrations of free (unbound) fatty acid in the medium, net fatty acid binding by either resting or activated T cells was considerably greater in the presence than in the absence of AFP; (4) in the presence of AFP, fatty acid binding was much higher in activated T cells than in resting T cells, whereas in the absence of AFP, nonsignificant differences were observed between activated and resting T cells; and (5) the time course of fatty acid and AFP binding at 4 degrees C revealed that, at equilibrium, the number of fatty acid molecules bound to the cell was much greater than that of AFP suggesting an accelerated dissociation of the fatty acid upon interaction of the AFP-fatty acid complex with putative cell receptors. It is concluded to the existence of an AFP/AFP-receptor pathway that facilitates the binding of fatty acids to T lymphocytes, particularly upon their blast transformation. This pathway may fulfill the increased requirement for fatty acids characteristic of proliferating cells and may serve to regulate the endocytosis of fatty acids with modulatory effects on lymphocyte function and to protect cells from their cytotoxic potential when internalized in excess.


Vasoactive intestinal peptide and forskolin regulate proliferation of the HT29 human colon adenocarcinoma cell line.

Although several lines of evidence implicate cAMP in the regulation of intestinal cell proliferation, the precise role of this second messenger in the control of the human colon cancer cell cycle is still unclear. In order to investigate the role of cAMP in HT29 cell proliferation, we have tested the effect of vasoactive intestinal peptide (VIP) and forskolin on DNA synthesis and cell number, focusing on the time-dependent efficacy of the treatment. The cells were arrested in G0/G1 phase by incubation for 24 h in serum-free medium and proliferation was re-initiated by addition of either 85 nM insulin or 0.5% fetal calf serum. In the presence of fetal calf serum, G1/S transition was found to occur earlier than with insulin. Exposure of the HT29 cells to 10(-5) M forskolin in the early stages of growth induction (within 12 h from FCS addition or within 14 h from insulin treatment) resulted in a significant inhibition of DNA synthesis and a delayed entry in the S phase. By contrast, VIP (10(-7) M) was inhibitory only when added within a narrow window (10 to 12 h or 12 to 14 h following FCS or insulin addition, respectively). The difference in efficiency of forskolin and VIP to inhibit cell proliferation may be correlated with their own potency to promote long-lasting cAMP accumulation. The combination of VIP plus forskolin had synergistic effects on both cAMP accumulation and cell-growth inhibition. Taken together, our data indicate that cAMP may act at a step in the late G1 or G1/S transition.


Common senescent cell-specific antibody epitopes on fibronectin in species and cells of varied origin.

The phenomenon of in vitro cellular senescence has been demonstrated in cultured cells derived from humans and various other species. We have previously shown that monoclonal antibodies SEN-1, SEN-2, and SEN-3 react to epitopes on fibronectin that are exposed when human diploid fibroblasts become senescent. We here present results demonstrating that exposure of these epitopes is specific to senescence for a variety of human cells: epidermal keratinocytes, mammary epithelial cells, as well as fibroblasts. Fibronectin from 11 additional species was also analyzed by Western immunoblot for ability to bind the SEN antibodies. SEN-1 bound only human and gorilla fibronectin, whereas SEN-2 and SEN-3 bound fibronectin from those two species as well as the horse, cow, sheep, goat, dog, and chick. None of the antibodies reacted with fibronectin from the rabbit, rat, or mouse. These data indicated a correlation between the ability of the SEN antibodies to bind fibronectin from a particular species and the ability of cells from that species to exhibit a stable senescent phenotype in vitro. Therefore, exposure of this region of fibronectin may be important in the establishment and maintenance of cellular senescence. In addition, the ability of the SEN antibodies to react with fibronectin from a variety of senescent cells emphasizes their usefulness as markers for cellular senescence.


Characterization of a novel Rochalimaea species, R. henselae sp. nov., isolated from blood of a febrile, human immunodeficiency virus-positive patient.

Isolation of a Rochalimaea-like organism from a febrile patient infected with human immunodeficiency virus was confirmed. Analysis of 16S rRNA gene sequences, together with polymerase chain reaction and restriction endonuclease length polymorphism analysis of a portion of the citrate synthase gene, demonstrated that the agent is closely related to members of the genus Rochalimaea and that the isolate is genotypically identical to the presumptive etiologic agent of bacillary angiomatosis. However, the same genotypic analyses readily differentiated the new isolate from isolates of other recognized Rochalimaea species as well as other genera of bacteria previously suggested as putative etiologic agents of bacillary angiomatosis and related syndromes. We propose that the novel species be referred to as Rochalimaea henselae sp. now.


Monoclonal antibodies and chemiluminescence immunoassay for detection of the surface protein of human T-cell lymphotropic virus.

Monoclonal antibodies (MAbs) raised against human T-cell lymphotropic virus type I (HTLV-I) recognized five distinct antigenic domains of viral env gene-encoded proteins. By using recombinant env proteins and synthetic peptides as mapping antigens, it was determined that the most immunogenic region represented a central portion of the retroviral surface protein (domain 2; amino acids 165 to 191). However, only a single MAb was able to react strongly with native viral proteins. This antibody (clone 6C2) was directed to an epitope within domain 4 (amino acids 210 to 306) of the retroviral env gene and reacted with envelope proteins in both HTLV-I and HTLV-II, as determined by immunoprecipitation, solid-phase binding, and immunoblotting. No reactivity against envelope components of other human retroviruses, including human immunodeficiency virus types 1 and 2, was present. Flow cytometry data demonstrated that MAb 6C2 reacted with cell lines chronically infected with HTLV-I or HTLV-II and also with surface antigens expressed on fresh adult T-cell leukemia cells, following up-regulation with interleukin-2. By a chemiluminescence immunoassay procedure, picogram amounts of viral surface protein could be detected in the unconcentrated supernatants of HTLV-infected cell lines and in diagnostic cultures. Levels of env and gag proteins released by cells into culture supernatants were not directly related to percent expression of cell surface viral-coat proteins. Further, the molar ratio of p19 to gp46 in conditioned media varied from strain to strain, possibly reflecting differences in viral assembly or packaging mechanisms. MAb 6C2 will be of value in characterizing the biochemical and immunological behavior of retroviral env gene proteins and in studying the interaction of HTLV-I and HTLV-II with their receptors.


Epidemiologic typing of Staphylococcus aureus by DNA restriction fragment length polymorphisms of rRNA genes: elucidation of the clonal nature of a group of bacteriophage-nontypeable, ciprofloxacin-resistant, methicillin-susceptible S. aureus isolates.

Analysis of DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) was employed to assist in the epidemiologic investigation of the emergence and spread of ciprofloxacin-resistant Staphylococcus aureus at the Atlanta VA Medical Center because many isolates of interest were nontypeable by phages and harbored few plasmids useful as strain markers. Chromosomal DNAs of selected S. aureus isolates were digested initially with 20 different restriction enzymes. EcoRI appeared to give the best discrimination of hybridization banding patterns (ribotypes) and was used with all study isolates. Overall, 15 different ribotypes were seen among the 50 S. aureus isolates studied (7 ribotypes among 13 methicillin-susceptible S. aureus [MSSA] isolates and 9 ribotypes among 37 methicillin-resistant S. aureus [MRSA] isolates). Seven of eight ciprofloxacin-resistant MSSA (CR-MSSA) patient isolates had identical antibiograms, were nontypeable by phages, and had a single 22-MDa plasmid. Six of these seven CR-MSSA isolates had an identical ribotype pattern. Ribotyping distinguished this CR-MSSA strain or clone from MRSA and other MSSA isolates, including nontypeable isolates that contained a 22-MDa plasmid. Five ciprofloxacin-susceptible MSSA isolates studied had five ribotypes; one pattern was identical to the CR-MSSA clone. Twenty-three CR-MRSA isolates recovered from the Atlanta VA Medical Center had four different ribotypes. Ribotyping proved to be a useful molecular epidemiologic tool in the study of S. aureus because it differentiated isolates which were indistinguishable by more traditional methods. In addition, this technique demonstrated that at our institution, ciprofloxacin resistance emerged in multiple strains of MRSA, as opposed to primarily a single strain or clone of MSSA.


Clinical and laboratory analyses of cytospin-prepared Gram stains for recovery and diagnosis of bacteria from sterile body fluids.

The smear of a clinical specimen provides essential laboratory information that is used to make therapeutic decisions. For this study, smears were made by centrifugation in a Beckman Microfuge 11 (Beckman Instruments, Palo Alto, Calif.) and in parallel by using a Cytospin 2 apparatus (Shandon Inc., Pittsburgh, Pa.). Of 350 consecutive body fluid specimens examined, 50 (14.0%) grew bacteria. Both methods were culture and smear positive for 24 (6.9%) specimens; 18 (5.1%) specimens were cytocentrifuge smear positive, culture positive, and high-speed centrifugation (HSC) negative; 3 (0.8%) were culture negative and positive by both smear methods; and 1 (0.2%) was HSC smear positive, culture positive, and cytocentrifuge negative. Seven (2.0%) specimens were culture positive and negative by both smear methods. Clinically, cytocentrifuge preparations showed greater sensitivity for culture-positive specimens and a closer correlation with the CFU per milliliter than HSC did, resulting in a greater ability to treat patients with specific therapies. In addition, analysts needed to examine only a 6-mm-diameter area on the slide, cells and microbes were somewhat larger and more regular in appearance, and smears stained more uniformly. Because of the increased clinical and laboratory utility of the cytocentrifuge, its use is recommended in clinical microbiology laboratories for all sterile body fluid specimens.


Pneumocystis carinii and specific fungi have a common epitope, identified by a monoclonal antibody.

Because Pneumocystis carinii may be related to fungi, we evaluated the reactivities of monoclonal antibodies raised against P. carinii with a variety of fungi. Fifty-two fungi and six protozoa were evaluated by immunofluorescence. One of three monoclonal antibodies (MAbs) tested (MAb 7D7) reacted with 15 fungi but no protozoa. Saccharomyces cerevisiae showed the strongest reactivity by immunofluorescence. The reactive antigen was characterized for four fungi by the immunoblot technique. In all cases the antigen that was reactive with MAb 7D7 was larger than the P. carinii antigens that reacted with 7D7. In further studies with P. carinii, Aspergillus species, and S. cerevisiae, we found that MAb 7D7 reacted with a carbohydrate component in all organisms. The presence of an epitope that is common to P. carinii and a number of fungi further supports the fungal nature of P. carinii.


Evidence for two serotype G3 subtypes among equine rotaviruses.

Ten cultivable equine rotavirus isolates, two of North American, six of British, and two of Irish origin, were compared with standard rotavirus strains and with each other by cross neutralization, neutralization with a panel of monoclonal antibodies (MAbs), hybridization to a simian rotavirus (SA-11) VP7 gene probe, and reaction with rotavirus subgrouping and serotyping MAbs in enzyme-linked immunosorbent assays. Six isolates, two of which had previously been serotyped as G3 by other workers, were found to be serotype G3; one was confirmed to be G5, and three were not related to serotypes G1 to G10. The serotype G3 strains were divisible into two subtypes, G3A and G3B, on the basis of cross neutralization. This division was also apparent in reactions with neutralizing VP7-specific MAbs and in the liquid hybridization assay. Two of the isolates were not bound by either subgroup MAb, six were bound by both subgroup I and II MAbs, and two were bound by only the subgroup I MAb. The assays used in this characterization provide a range of epidemiological information for use in future field investigations.


Local immune responses to the Campylobacter flagellin in acute Campylobacter gastrointestinal infection.

Intestinal immunoglobulin A (IgA) anti-campylobacter flagellin antibodies were measured by an enzyme-linked immunosorbent assay in patients with and without campylobacter infections. The level of fecal IgA antiflagellin antibodies was significantly higher in patients than in controls, but only 12 of 29 patients (41.4%) with a campylobacter infection had detectable IgA antibodies against flagellin (optical density, greater than or equal to 0.100). Further testing of fecal IgA antibodies from 10 patients with homologous and heterologous purified flagellins showed strain specificity in 1 patient. Cross-reactivity of fecal IgA antibodies with heterologous flagellins did not correlate with the Lior serotype of the isolate.


Intracellular events involved in CD5-induced human T cell activation and proliferation.

In this report we describe a novel pathway of human T cell activation and proliferation involving the CD5 surface Ag. The CD5-specific Cris1 mAb induces by itself monocyte-dependent proliferation of PBMC. Among a panel of CD5-specific mAb (Leu1, OKT1, LO-CD5a, F101-1C5, and F145GF3), only the F145GF3 mAb shared this property with Cris1. The analysis of the biochemical pathway involved in this activation showed the lack of detectable hydrolysis of inositol phosphates or early increments in the intracellular cytosolic calcium concentration, after triggering cells with the mitogenic CD5 mAb. However, stimulation with CD5 induces activation of protein kinase C, as measured by phosphorylation of a specific peptide substrate (peptide GS), which can be inhibited by a pseudosubstrate peptide inhibitor. Stimulation with CD5 mAb induces also tyrosine kinase activity, with a substrate pattern that differs from that induced after triggering lymphocytes through the TCR-CD3 complex. On the other hand the IL-2/IL-2R pathway seems involved in the CD5-mediated proliferation of PBMC because anti-IL-2R-specific mAb inhibits CD5-induced proliferation, and stimulation with mitogenic CD5 mAb induces production of IL-2 and expression of IL-2R alpha and beta chains. Therefore, the triggering of the CD5 Ag can induce IL-2- and monocyte-dependent human T cell proliferation by a biochemical pathway that differs, at least in the first stages, from the one that mediates TCR-CD3 complex-induced T cell activation.


Gene transfer demonstrates that the V gamma 1.1C gamma 4V delta 6C delta T cell receptor is essential for autoreactivity.

Murine T cell lines and hybridomas derived from the epidermis that express the V gamma 1.1C gamma 4V delta 6C delta TCR and may, therefore, recognize an autoantigen, secrete cytokines spontaneously in culture. In addition, activation of these cells requires engagement of the vitronectin receptor (VNR) by extracellular matrix proteins. To further evaluate the role of the TCR, the VNR, and the putative autoantigen in the activation of this T cell subset, we cloned complete cDNA encoding the V gamma 1.1C gamma 4 and V delta 6C delta TCR and transfected the cDNA constructs into a TCR- murine hybridoma and into a TCR- variant of the human Jurkat line. The murine transfectant spontaneously produced IL-2 in culture and IL-2 production could be inhibited by anti-CD3, anticlonotypic mAb to the transfected TCR, and anti-VNR mAb, as well as by RGDS. These results demonstrate that transfection of the gamma delta TCR confers to recipient T cells the phenotype of constitutive activation, as well as dependence on engagement of the VNR as an accessory molecule. In contrast, the Jurkat gamma delta transfectant failed to produce cytokines spontaneously, although the transfected TCR was capable of signal transduction after stimulation by anti-TCR mAb. Surprisingly, neither the murine transfectant nor the human transfectant could be induced to respond to autoantigen bearing cells in coculture assays. One interpretation of these results is that coexpression on the surface of the same cell of the V gamma 1.1 V delta 6 TCR, the VNR, and a putative autoantigen are necessary for T cell activation in this system.


Monoclonal antibodies directed to different epitopes in the CD3-TCR complex induce different states of competence in resting human T cells.

After the initial stages of activation, T cells are not able to proliferate on their own but become competent to proliferate in response to exogenously added lymphokines. In the present study we compared the capacity of mAb directed to CD3 (OKT3, Leu4, UCHT1) or to common epitopes on the alpha/beta T-cell receptor (BMA 031, BMA 032) to induce competence in purified resting T cells. Stimulation with either soluble anti-CD3 or anti-alpha/beta TCR mAb rendered cells competent to progress to DNA synthesis in response to exogenous IL-2. In contrast, only soluble BMA 031 and BMA 032 were able to induce responsiveness to IL-4; anti-CD3 mAb had either to be immobilized or used in combination with anti-CD28 mAb to induce responsiveness to IL-4. Further, BMA 031-induced IL-4 responsiveness was selectively found in the CD45RA+ T cell subset. Analysis of early activation events revealed that the capacity of soluble BMA 031 and BMA 032 to induce responsiveness to IL-4 did not correlate with the ability of these mAb to increase the level of cytosolic Ca2+ or to induce detectable tyrosine phosphorylation. On the other hand, soluble Leu4 (anti-CD3) triggered an increase in both intracellular Ca2+ and tyrosine phosphorylation but was unable to induce IL-4 responsiveness. These data indicate that the induction of IL-2 and IL-4 responsiveness requires different sets of activation signals which can be induced by stimulating different epitopes in the CD3-TCR complex. This supports the concept that distinct activation pathways are coupled to the CD3-TCR complex.


Diversity in fine specificity and T cell receptor usage of the human CD4+ cytotoxic T cell response specific for the immunodominant myelin basic protein peptide 87-106.

Multiple sclerosis (MS), a human demyelinating disease, is thought to be caused by an autoimmunologic process, and myelin basic protein (MBP) is considered a likely autoantigen. Studies of T cell lines (TCL) responding to different parts of the MBP molecule have indicated that amino acids 87 through 106 contain an immunodominant epitope of MBP. We have demonstrated previously that amino acids 89 through 99 represent the core of this 87-106 peptide epitope. Importantly, this epitope is not only encephalitogenic in SJL/J mice and Lewis rats but also has been shown to be recognized by human cytotoxic TCL in the context of four HLA-DR molecules that are associated with MS in different geographic areas. If the immune response to MBP peptide 87-106 was homogeneous with respect to epitope specificity and TCR usage, specific immunotherapies targeting the interaction of peptide, MHC, and TCR might be possible. In this study, the fine specificity of 29 CD4+ cytotoxic, long term, and limiting dilution TCL that had been generated against whole MBP and were derived from four MS patients and two healthy relatives was dissected using truncated and alanine-substituted peptides for the 87-106 peptide. In addition, the TCR alpha and beta chain usage of 15 CD4+ TCL was determined. Using truncated peptides, the presence of several nested immunogenic epitopes within amino acids 87 to 106 was demonstrated. TCL with identical restriction elements and similar responses to truncated peptides could be differentiated further using alanine-substituted peptides. Finally, heterogeneity of TCR usage was shown not only for those lines that differed in their peptide specificity but also for some that showed identical responses and were restricted by the same HLA-DR antigen. In conclusion, the CD4+ cytotoxic T cell response to the immunodominant MBP peptide 87-106 demonstrates a high degree of heterogeneity at the level of fine specificity and TCR usage. These findings indicate that specific immunotherapies aimed at TCR in MS will probably be more complicated than previously anticipated.


Evidence for recent as well as long term activation of T cells migrating through endothelial cell monolayers in vitro.

As T cells actively extravasate from blood, they adhere to endothelium and then migrate out of the vessel with a locomotive activity. Although both adhesion and locomotion are properties associated with activated T cells, the two processes are not necessarily associated with identical activation states. Using human endothelial cells (EC) cultured to confluence on collagen gel, we examined the activation state of human peripheral blood T cells that adhere to and migrate through EC monolayers with three different methods: flow cytometric analysis of cell surface activation-related molecules, incorporation of tritiated nucleotide, and cell cycle analysis. The results were as follows. 1) Although expression of very late activation Ag integrins VLA-2 and VLA-3 by the initial blood T cell population (unseparated cells) and of adherent T cells was minimal, 40 to 45% of migrating cells were positive for VLA-2 and VLA-3. 2) The percentage of IL-2R+ cells in both unseparated and adherent cells was below 5% whereas the percentage of IL-2R+ cells among the migrating cells was 22 +/- 9% (range, 12 to 31%, n = 6). 3) Migrating cells expressed the highest CD26, whereas CD26 of adherent (nonmigrating) cells was divided into negative and high expression; in contrast, leukocyte adhesion molecule-1 (L-selectin) of both adherent and migrating cells was mostly low or negative. 4) [3H]Uridine incorporation of migrating and adherent cells was 2.1- to 2.5-fold and 1.4- to 1.7-fold higher, respectively, than that of unseparated cells, indicating that RNA synthesis of migrating cells as well as adherent cells was enhanced. 5) Cell cycle analysis showed that 23.5% of migrating cells appeared to enter the G1 phase but not S or G2 + M phases whereas 2.2% of unseparated cells and 8.0% of adherent cells that did not migrate had an RNA content consistent with entry into G1. These results suggest that cells migrating from normal human blood through unactivated EC have been activated recently as well as showing evidence of long term activation. The activation state of migrating cells is consistent with the hypothesis that previous in vivo activation is required for cells to migrate through EC in this system.


Synthetic peptides anchor T cell-specific TNP epitopes to MHC antigens.

Several TNP-specific, H-2Kb-restricted mouse CTL clones were identified which specifically lysed target cells in the presence of tryptic digests of TNP-modified BSA. Glutaraldehyde fixation of cells revealed that the tryptic fragments did not require further cellular processing. Chromatographic fractionation of digested TNP-BSA identified the peptide TNP-BSA222-231, containing a TNP-modified lysine at BSA position 227, as the antigenic entity. The corresponding synthetic peptide was immunologically cross-reactive with the digest. All clones reactive with TNP-BSA222-231 cross-reacted with a similar peptide from mouse serum albumin (TNP-MSA126-135), favoring the assumption that TNP-BSA222-231 represents an artificial determinant, cross-reacting with some as yet unidentified, TNP-modified, Kb-associated self-peptides. Some of our clones also cross-reacted with tryptic digests of TNP-OVA or TNP-keyhole limpet hemocyanin. We interpret these findings to indicate that 1) a significant proportion of hapten (TNP) determinants for T cells are anchored to MHC via peptides; and 2) the amino acid sequence of these peptides may only partly define the specificity of the T cell-relevant hapten epitope, implying a particularly repetitive nature of these determinants. The production of T cell-antigenic hapten-peptide conjugates will hopefully open new roads to study immune responses to environmental allergens.