Outcome of children born to women treated during pregnancy for the antiphospholipid syndrome. To determine the rate of neonatal and childhood medical complications in the offspring of women with the antiphospholipid syndrome who are treated during pregnancy. We compared 29 infants born to 23 mothers with antiphospholipid syndrome with a group of control children matched for year and gestational age at birth and route of delivery. During pregnancy, mothers with antiphospholipid syndrome were treated with accepted therapeutic regimens, including prednisone and low-dose aspirin, heparin and low-dose aspirin, and others. Neonatal complications in the study infants included hyperbilirubinemia (14), respiratory distress syndrome (three), bronchopulmonary dysplasia (two), necrotizing enterocolitis (two), intraventricular hemorrhage (two), neonatal sepsis (one), coarctation of the aorta (one), hypothyroidism (one), hypoglycemia (one), and death (one). Two children had feeding problems, four had delayed psychomotor development, and eight were small for their age. However, the overall rate of neonatal or childhood complications did not differ from that in the control children. Our data indicate that prematurity and neonatal problems are common, but the childhood course for these offspring is similar to that of other premature infants.


Microwave-stimulated chemical fixation of whole eyes. Microwave-stimulated chemical fixation has been used successfully to rapidly prepare nonocular tissue for processing and paraffin embedding. This same technique was adapted to fix whole eyes using a common domestic microwave oven. Histologic sections were compared with sections from globes that were fixed in a traditional manner. Histologic sections and histochemical and immunohistochemical staining of eyes treated with microwave-stimulated chemical fixation in 10% neutral-buffered formalin were comparable in quality to eyes fixed in 10% neutral-buffered formalin at room temperature for 48 hours. Because microwave irradiation accelerates the most time-consuming part of preparing whole eyes for light microscopy, the technique enhances laboratory efficiency. The technique is particularly well suited for ensuring adequate fixation of globes when histologic diagnosis is needed in less than 48 hours.


Developing shared decision-making programs to improve the quality of health care. We strongly believe in the importance of patient involvement in a medical decision. The interactive SDPs appear to be an effective way to facilitate this involvement. One key to the acceptance of these programs by patients and physicians is that they be--and be perceived as--fair, accurate, and balanced. Herein we have described the well-defined protocol for developing, evaluating, and updating SDPs. The first of the foundation's programs dealing with benign prostatic hyperplasia has been well received by patients and clinicians and has been demonstrated to have an impact on practice patterns. Efforts are under way to evaluate four additional programs, leading to widespread availability of the first five SDPs by fall of 1992.


Biochemistry of migraine.

Biochemistry of migraine. The biochemistry of migraine is complex. Many contradictory or never replicated findings in often small patient groups have been published. The following observations in the platelet-free plasma and urine appear to have some solid basis and will be discussed: 1) systemic derangement of 5-HT metabolism, relevant to the peripheral vascular component of migraine pathophysiology, 2) changes in neuroexcitatory amino acids and magnesium, which may reflect a predisposition of the migraine patient, notably those having attacks with aura, to develop spreading depression, 3) alterations in methionine-enkephalin levels, which may be a useful marker to discriminate between tension headache and migraine, 4) hormonal fluctuations which seem important to set the threshold for an attack, 5) changes of vasoactive peptides in the cranio-vascular circulation, providing the first human evidence that the trigemino-vascular system indeed is relevant in migraine, and 6) catecholaminergic changes suggesting sympathetic overactivity. Finally distinct biochemical differences between patients with migraine without aura and patients with tension headache on one hand, and between patients with migraine with aura and patients with migraine without aura on the other hand will be emphasized. Findings in platelets will be discussed only if they are complementary and supportive to the plasma and urine data.


The trigemino-vascular system and migraine. Neurogenic inflammation has been proposed as a possible pathogenetic mechanism for migraine and cluster headache. Antidromic stimulation of trigeminal fibers causes plasma protein extravasation, mast cell activation and degranulation, vacuolation and increase in endothelial vesicle number within post capillary venules in rat dura mater. The antimigraine drugs sumatriptan and dihydroergotamine block the development of plasma extravasation and ultrastructural changes, as well as plasma calcitonin gene-related peptide (CGRP) increase in the superior sagittal sinus following electrical trigeminal ganglion stimulation. Sumatriptan and dihydroergotamine bind with high affinity to the 5-HT1D/1B receptors, thus suggesting that their neurogenic antiinflammatory activity is mediated by activation of 5-HT autoreceptors present on sensory fibers innervating blood vessels in dura mater.


The pathophysiology of migraine: a tentative synthesis. It is proposed that the migraine attack is explicable by an interaction between the brain and the cranial circulation in subjects with unstable vascular and pain-control mechanisms. While peptides are undoubtedly involved in vasodilatation, there is strong evidence that 5-HT plays an important part in the genesis of migraine. Whether the place of 5-HT lies in central pain-control pathways, in the serotonergic project to the cerebral cortex, in a direct action on the cranial blood vessels or in its action at all three sites remains uncertain. It seems probable that the primary action of sumatriptan or ergotamine in terminating migraine headache is exerted on the cerebral and extracranial circulation whereas medications employed in prophylaxis may act centrally.


Fine needle aspiration biopsy cytology as an adjunct in the diagnosis of childhood sarcoidosis. Fine needle aspiration biopsy cytology performed in three children with sarcoidosis expedited clinical investigation and diagnosis of their disease. Each patient had a different clinical presentation; in two of them lymphoma was part of the initial differential diagnosis. Aspiration cytology in all cases revealed collections of epithelioid histiocytes, and multinucleate foreign body-type giant cells, without accompanying necrosis or acute inflammation. A diagnosis of non-caseating granulomas consistent with sarcoidosis was made in all aspirates. Special stains for identification of organisms performed on the smears of one case, and culture of aspirate material from one case were negative. Subsequent serum angiotensin converting enzyme levels in all patients were elevated. Chest x-ray films in all patients showed mediastinal and hilar lymphadenopathy. One patient had an interstitial pulmonary infiltrate. All patients responded to steroid therapy. Fine needle aspiration biopsy can be a useful diagnostic tool in the evaluation of children with suspected sarcoidosis.


Comparative investigations of the morphology and chemical composition of the eggshells of Acanthocephala. II. Palaeacanthocephala. Eggshells of the palaeacanthocephalans Acanthocephalus anguillae, Pomphorhynchus laevis and Polymorphus minutus were investigated for their fine structure as well as their chemical composition. The acanthor larvae are surrounded by four eggshells (E1-E4) separated by interstices (G1-G4). Immature eggs do not exhibit the complete set of eggshells. The chemical composition of the outermost, thin eggshell (E1) remains unknown. E2 is supplied by outer filaments of different strength; it contains keratin, which was localized electron microscopically using anti-keratin. In P. laevis and P. minutus, E3 seems to contain glycoproteins, which could not be visualized in this eggshell of A. anguillae. The innermost eggshell (E4) uniformly contains chitin. The electron-lucent interstices of the eggs of P. laevis and P. minutus are rich in polysaccharides and/or proteoglycans, whereas those of A. anguillae contain low amounts of such substances. The differences observed in the abundance of carbohydrates are discussed with respect to the life cycles of the three acanthocephalans.


A comparison of sporozoite and cyst merozoite surface proteins of Sarcocystis. Surface labeling of Sarcocystis muris and S. suicanis sporozoites with N-hydroxysuccinimide biotin led to the detection of major membrane proteins with relative molecular weights of 29 and 30 kDa, respectively. Immunoblots of Sarcocystis sporozoite proteins probed with sera from infected hosts or with polyclonal monospecific antibodies generated against membrane antigens of cyst merozoites (noncorresponding stages) showed cross-reactivity between the two developmental stages (cyst merozoites and sporozoites) as well as between the species S. muris and S. suicanis. Two-dimensional gel electrophoresis resulted in the identification of isoforms of the sporozoite membrane antigens, with isoelectric points ranging from pH 4.7 to pH 6.4 for S. muris and from pH 4.7 to pH 5.2 for S. suicanis. The molecular masses, the charge heterogeneity, and the immunological reactivity of the surface proteins of Sarcocystis sporozoites were similar to those of cyst merozoites of both species.


Conditions affecting the immunohistochemical detection of HIV in fixed and embedded renal and nonrenal tissues. A number of studies have suggested that HIV infection can be detected in a variety of routinely fixed archival tissues using antibodies to various viral proteins. In order to study this immunocytochemical approach, paraffin sections were examined with a large panel of commercially available monoclonal antibodies against the various HIV proteins (5 antibodies to p24, 1 to p17, 1 to gp41, and 1 to gp120) using a streptavidin-biotin method. A polyclonal antibody against p24 was also tested. Formalin-fixed, paraffin-embedded HIV infected CEM E5 T cells were used as positive controls. Tissues from AIDS patients included 31 kidneys, 8 lymph nodes, 2 spleens and 3 brains. Non-AIDS tissues examined were 6 renal biopsies with focal segmental glomerulosclerosis, 5 with interstitial nephritis, 6 reactive lymph nodes, and a brain with encephalitis, all from patients not known to be at high risk for HIV infection. Additional negative controls included: 1) replacement of primary antibody with a hybridoma derived mouse monoclonal IgG1 standard, 2) omission of the primary antibody, and 3) sections of formalin-fixed paraffin-embedded CEM E5 T cells cultures not infected with HIV. Competition experiments with excess recombinant p24 protein were also performed. False positive staining with the IgG1 standard or with the antibodies to HIV proteins was frequently seen in tissues with pathologic findings (inflammation, hyalin degeneration), particularly following protein digestion. Protein digestion also had a major impact on specific staining. Digestion with proteinase K abolished specific staining for the core proteins of the virus (p17, p24) on the positive control sections.(ABSTRACT TRUNCATED AT 250 WORDS)


[Correlation between the nucleolar organizer region in adenocarcinoma of the prostate and the Gleason system]. Using a silver staining, technique, Nucleolar Organizer Region-associated proteins (NORs) were studied on paraffin sections of 25 resected prostatic adenocarcinomas classified with Gleason grading and 11 hyperplastic lesions. Then 7 inclusions was selected for each grade of Gleason system and 7 inclusions of normal prostatic tissue. The mean numbers of argyrophilic nucleolar organizer regions (AgNORs) increased significantly (P less than 0.01) from normal prostatic tissue to Gleason 5. The data indicate that AgNORs counts may help distinguish between each grade of Gleason system. It was concluded that the AgNOR technique provides a significant kinetic evaluation of prostatic adenocarcinoma and its prognostic study.


Chromosomes in the diagnosis of soft tissue tumors. I. Synovial sarcoma. It has been established that nonrandom chromosome rearrangements are characteristic of specific types of neoplasia. We present six new cases of sarcoma that had in common the same chromosome abnormality, i.e., a balanced translocation between chromosomes X and 18, t(X;18)(p11.2;q11.2), and evaluate the 15 cases with this translocation in the literature. The histological diagnosis was synovial sarcoma in 19 cases and malignant fibrous histiocytoma and fibrosarcoma in the remaining two tumors, respectively. The translocation was found in tumors of both the biphasic and monophasic types, as well as in poorly differentiated synovial sarcoma. The two nonsynovial sarcomas with the t(X;18) were described as spindle cell tumors but failed to show the presence of cytokeratins by immunohistochemical stains. Even with the numerous variabilities on which this test depends, the cytogenetic analysis holds great promise as a tool for the diagnosis of synovial sarcoma.


Lymphoid cells transformed by Abelson virus require the v-abl protein-tyrosine kinase only during early G1. Cells infected with temperature-sensitive transformation mutants of the Abelson murine leukemia virus express low levels of kinase activity at the nonpermissive temperature, causing transformed pre-B cells to die under these conditions. Examination of cell cycle profiles of such populations prior to cell death reveals that the cells accumulate in the G1 phase of the cell cycle. Following G1 arrest, the cells die via apoptosis, an active process of cell elimination. Cell synchronization and temperature-shift experiments show that G1 arrest reflects the requirement for a functional v-abl protein during early G1 and that the molecule is not required at other phases of the cell cycle. These data indicate that the substrate(s) critical to v-abl-mediated transformation is involved in regulating G1 transit and that these interactions are dominant over all other changes required for the multistep process that results in the fully malignant phenotype associated with v-abl expression in lymphoid cells.


Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme. Nitric oxide (NO) is a messenger molecule of macrophages, endothelial cells in blood vessels, and neurons. A neuronal form of NO synthase (NOS) has been previously cloned. We now report the molecular cloning of macrophage NOS. The macrophage enzyme displays 50% sequence identity to the neuronal enzyme. Like neuronal NOS, macrophage NOS has recognition sites for FAD, FMN, and NADPH and also has a consensus calmodulin binding site. Macrophage NOS mRNA is strikingly inducible; it is absent in quiescent macrophages or spleen but is prominent 2-6 hr after endotoxin treatment.


Cloning and expression of a Ca(2+)-inhibitable adenylyl cyclase from NCB-20 cells. A cDNA that encodes an adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] has been cloned from NCB-20 cells, in which adenylyl cyclase activity is inhibited by Ca2+ at physiological concentrations. The cDNA clone (5.8 kilobases) was isolated by polymerase chain reaction (PCR) using degenerate primers designed by comparison of three adenylyl cyclase sequences (types I, II, and III) and subsequent library screening. Northern analysis revealed expression of mRNA (6.1 kilobases) corresponding to this cDNA in cardiac tissue, which is a prominent source of Ca(2+)-inhibitable adenylyl cyclase. The clone encodes a protein of 1165 amino acids, whose hydrophilicity profile was very similar to those of other mammalian adenylyl cyclases that have recently been cloned. A noticeable difference between this protein and other adenylyl cyclases was a lengthy aminoterminal region before the first transmembrane span. Transient expression of this cDNA in the human embryonic kidney cell line 293 revealed a 3-fold increase in cAMP production in response to forskolin compared with control transfected cells. In purified plasma membranes from transfected cells, increased adenylyl cyclase activity was also detected, which was susceptible to inhibition by submicromolar Ca2+. Thus, this adenylyl cyclase seems to represent the Ca(2+)-inhibitable form that is encountered in NCB-20 cells, cardiac tissue, and elsewhere. Its identification should permit a determination of the structural features that determine the mode of regulation of adenylyl cyclase by Ca2+.


Cloning and expression of a cAMP-activated Na+/H+ exchanger: evidence that the cytoplasmic domain mediates hormonal regulation. The ubiquitous plasma membrane Na+/H+ exchanger (termed NHE1) is activated by diverse hormonal signals, with the notable exception of hormones acting through cAMP as second messenger. Therefore, the Na+/H+ exchanger found in the nucleated trout red cell is of particular interest since it is activated by catecholamines, forskolin, and cAMP analogues. We report here that a cloned cDNA encoding the red cell exchanger restores functional Na+/H+ activity when transfected into Na+/H+ antiporter-deficient fibroblasts (i.e., it regulates intracellular pH in a Na-dependent and amiloride-sensitive manner). This red cell exchanger represents an additional form of Na+/H+ exchanger (termed beta NHE), which is characterized by a specific cytoplasmic domain involved in activation by the cAMP-dependent signaling pathway. After transfection in the same cellular context, beta NHE, but not NHE1, is activated by cAMP or by hormones that increase cAMP levels. Comparison of the amino acid sequences of exchangers shows that beta NHE, but not NHE1, contains two clustered consensus motifs for phosphorylation by a cAMP-dependent protein kinase (protein kinase A; PKA). A deletion mutant devoid of the C-terminal region of the cytoplasmic loop containing the two PKA sites restores Na+/H+ activity but is no longer activated by cAMP analogues or catecholamines. In red blood cells, the Na+/H+ exchanger is also activated by another pathway involving protein kinase C (PKC). Expression of beta NHE in fibroblasts shows that these two independent signaling pathways impinge on two distinct domains of the exchanger. The cytoplasmic segment containing PKA consensus sites, which is crucial for cAMP activation, is unnecessary for stimulation by PKC activators.


The partially folded conformation of the Cys-30 Cys-51 intermediate in the disulfide folding pathway of bovine pancreatic trypsin inhibitor. The best-characterized protein folding pathway is that of bovine pancreatic trypsin inhibitor, which folds from the reduced form through a series of disulfide bond intermediates. The crucial one-disulfide intermediate of bovine pancreatic trypsin inhibitor with the disulfide bond between Cys-30 and Cys-51 is shown here to have a partially folded conformation in which the major elements of secondary structure interact via a core of apolar side chains, which resembles part of the native conformation. The stability of this structure can account for the predominance of this one-disulfide intermediate during folding. Much of the remaining one-third of the polypeptide chain, in particular the N-terminal 14 residues, is largely disordered; this accounts for the ability of this intermediate to form readily any of the three possible second disulfide bonds involving Cys-5, -14, and -38. The partially folded conformation of this intermediate provides direct evidence for the importance of native-like interactions between elements of secondary structure in directing protein folding, which is assumed in many studies.


Antisense oligodeoxynucleotides to the cystic fibrosis transmembrane conductance regulator inhibit cAMP-activated but not calcium-activated chloride currents. Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cAMP-dependent protein kinase leads to chloride flux in epithelial cells. Is CFTR also required for the calcium-dependent activation of chloride channels? We used antisense oligodeoxynucleotides to CFTR to reduce the expression of CFTR in colonic and tracheal epithelial cells. The antisense oligomers were a pair of adjacent 18-mers complementary to nucleotides 1-18 and 19-36 of CFTR mRNA. Sense and misantisense oligomers served as controls. A 48-h antisense treatment reduced the expression of CFTR protein as assayed by immunoprecipitation and autoradiography to 26% of the level in sense-treated T84 cells. Whole-cell patch clamp revealed that a 48-h antisense treatment of T84 and 56FHTE-8o- fetal tracheal epithelial cells reduced the cAMP-activated chloride current to approximately 10% of that in sense-treated cells. The half-life of functional CFTR is less than 24 h in these cells. In contrast, the calcium-activated chloride current was not affected by antisense treatment. Hence, the cAMP and calcium pathways are separate. CFTR is required for the cAMP pathway but not for the calcium pathway.


Presence of a member of the Tc1-like transposon family from nematodes and Drosophila within the vasotocin gene of a primitive vertebrate, the Pacific hagfish Eptatretus stouti. Molecular cloning of the vasotocin gene of a cyclostome, the Pacific hagfish Eptatretus stouti, reveals, in contrast to other known members of the vertebrate vasopressin/oxytocin hormone gene family, an unusual exon-intron organization. Although the location of three exons and two introns is conserved, an additional intron is present 5' of the coding region of the hagfish gene. The third intron, which is greater than 14 kilobase pairs in size, contains on the opposite DNA strand to that encoding vasotocin an open reading frame exhibiting striking similarity to the putative transposase of Tc1-like nonretroviral mobile genetic DNA elements, so far reported only from nematodes and Drosophila. The hagfish element, called Tes1, is flanked by inverted terminal repeats representing an example of the existence of a typical inverted terminal-repeat transposon within vertebrates. The presence of Tc1-like elements in nematodes, Drosophila, and cyclostomes indicates that these genetic elements have a much broader phylogenetic distribution than hitherto expected.


The yeast nuclear gene suv3 affecting mitochondrial post-transcriptional processes encodes a putative ATP-dependent RNA helicase. Mitochondrial gene expression is controlled largely through the action of products of the nuclear genome. The yeast nuclear gene suv3 has been implicated in a variety of mitochondrial posttranscriptional processes and in translation and, thus, represents a key control element in nuclear-mitochondrial interactions. We have exploited a property of a mutant allele of suv3, SUV3-1, that causes, among other effects, a massive increase in the abundance of excised group I introns to clone the wild-type gene by a strategy of colony Northern hybridization. We have determined that the 84-kDa deduced protein product of the suv3 gene, which maps to chromosome XVI, has a typical mitochondrial targeting presequence and additional sequence motifs that suggest that it belongs to a family of ATP-dependent RNA helicases, enzymes whose importance in post-transcriptional and translational events has recently become apparent. We have identified the SUV3-1 mutation as a G----T transversion that creates a Val----Leu substitution in a 10-amino acid block that is highly conserved among ATP-dependent RNA helicases. We discuss some implications of this mutation on the effects of the SUV3-1 allele on mitochondrial RNA metabolism.


Cone visual pigments are present in gecko rod cells. The Tokay gecko (Gekko gekko), a nocturnal lizard, has two kinds of visual pigments, P467 and P521. In spite of the pure-rod morphology of the photoreceptor cells, the biochemical properties of P521 and P467 resemble those of iodopsin (the chicken red-sensitive cone visual pigment) and rhodopsin, respectively. We have found that the amino acid sequence of P521 deduced from the cDNA was very similar to that of iodopsin. In addition, P467 has the highest homology with the chicken green-sensitive cone visual pigment, although it also has a relatively high homology with rhodopsins. These results give additional strength to the transmutation theory of Walls [Walls, G. L. (1934) Am. J. Ophthalmol. 17, 892-915], who proposed that the rod-shaped photoreceptor cells of lizards have been derived from ancestral cone-like photoreceptors. Apparently amino acid sequences of visual pigments are less changeable than the morphology of the photoreceptor cells in the course of evolution.


Increasing binding affinity of agonists to glutamate receptors increases synaptic responses at glutamatergic synapses. This study examined the relationship between the affinity of glutamate agonists for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and the characteristics of the physiological responses elicited by endogenous activation of the AMPA receptors. We tested the effects of chaotropic ions on [3H]AMPA binding in synaptic membranes as well as on synaptic responses elicited in CA1 by electrical stimulation of the Schaffer/commissural pathway in the in vitro hippocampal slice preparation. Of the chaotropic ions tested, only perchlorate and thiocyanate produced large increases in [3H]AMPA binding to synaptic membranes. The effect was due to an increase in affinity for agonists, as shown by a shift of the displacement curves of 6-cyano-7-nitro[3H]-quinoxaline-2,3-dione binding by AMPA or glutamate. The effect of thiocyanate on [3H]AMPA binding was extremely sensitive to temperature, as the binding was increased almost 10-fold at 0 degree C but only 2- to 3-fold at 35 degrees C. The effect of perchlorate was only weakly temperature dependent. Similarly, thiocyanate and perchlorate were the only chaotropic ions tested that increased the initial slope and amplitude of the extracellularly recorded potentials evoked in CA1 dendritic field. Both ions did not change paired-pulse facilitation, an index of transmitter release, or fiber volley amplitude, an index of afferent recruitment. The chaotropic ions had no significant effects on either [3H]glutamate binding to the N-methyl-D-aspartate receptor or N-methyl-D-aspartate receptor-mediated synaptic responses. Finally, the effect of perchlorate on synaptic responses was significantly reduced after induction of long-term potentiation. These results indicate that an increase in affinity of the AMPA receptors for their agonists results in increased synaptic responses and strongly suggest that characteristics of the AMPA receptor are modified following long-term potentiation.


Large induction of keratinocyte growth factor expression in the dermis during wound healing. Recent studies have shown that application of basic fibroblast growth factor (basic FGF) to a wound has a beneficial effect. However, it has not been assessed whether endogenous FGF also plays a role in tissue repair. In this study we found a 160-fold induction of mRNA encoding keratinocyte growth factor (KGF) 1 day after skin injury. This large induction was unique within the family of FGFs, since mRNA levels of acidic FGF, basic FGF, and FGF-5 were only slightly induced (2- to 10-fold) during wound healing, and there was no expression of FGF-3, FGF-4, and FGF-6 detected in normal and wounded skin. High levels of FGF receptor 1 and FGF receptor 2 mRNA and low levels of FGF receptor 3 mRNA were found in both normal and wounded skin. No change in the levels of these transcripts was detected during wound healing. In situ hybridization studies revealed highest levels of KGF mRNA expression in the dermis at the wound edge and in the hypodermis below the wound. In contrast, mRNA encoding the receptor of this growth factor (a splice variant of FGF receptor 2) was predominantly expressed in the epidermis. These results suggest that basal keratinocytes are stimulated by dermally derived KGF during wound healing and implicate a unique role of this member of the FGF family in wound repair.


Transgenic mice expressing a mutant keratin 10 gene reveal the likely genetic basis for epidermolytic hyperkeratosis. Epidermolytic hyperkeratosis (EH; previously called bullous congenital ichthyosiform erythroderma) is an autosomal dominant skin disease of unknown etiology, affecting approximately 1 out of 300,000 people. It is typified by hyperkeratotic scaliness, blistering due to cytolysis within suprabasal epidermal cells, and hyperproliferation in basal cells. Histologically, EH epidermis exhibits a thickened stratum corneum and granular layer, with enlarged and irregular-shaped cells. Ultrastructurally, only suprabasal layers are affected, with three major aberrancies: (i) tonofilament clumping, (ii) nuclei and keratohyalin granules of irregular shape and size, and (iii) cell degeneration. We have discovered that transgenic mice expressing a mutant keratin 10 gene have the EH phenotype, thereby suggesting that a genetic basis for human EH residues in mutations in genes encoding suprabasal keratins K1 and K10. In addition, we show that (i) stimulation of basal cell proliferation can arise from a defect in suprabasal cells, and (ii) distortion of nuclear shape or aberrations in cytokinesis can occur when an intermediate filament network is perturbed.


Unequal human immunodeficiency virus type 1 reverse transcriptase error rates with RNA and DNA templates. Sequence variation in the type 1 human immunodeficiency virus (HIV-1) results, in part, from inaccurate replication by reverse transcriptase. Although this enzyme is error-prone during synthesis in vitro with DNA templates, the fidelity of RNA-dependent DNA synthesis relevant to minus-strand replication in the virus life cycle has not been examined extensively. In the present study, we have developed a system to determine the fidelity of transcription and reverse transcription and have used it to compare the fidelity of DNA synthesis by the HIV-1 reverse transcriptase with RNA and DNA templates of the same sequence. Overall, fidelity was several-fold higher with RNA than with DNA. Sequence analysis of mutants generated with the two substrates revealed that differences in error rates were substantial for specific errors. Fidelity with RNA was greater than 10-fold higher for substitution and minus-one nucleotide errors at five different homopolymeric positions. Because such errors likely result from template-primer slippage, this result suggests that misaligned intermediates are formed and/or used less frequently with an RNA template-DNA primer than with a DNA template-DNA primer. The results also suggest that HIV-1 reverse transcriptase synthesis with an RNA template-DNA primer was error-prone during incorporation of the first two nucleotides, perhaps due to aberrant enzyme-substrate interactions as synthesis initiates. The unequal error rates with RNA and DNA templates suggest that mistakes during minus- and plus-strand DNA synthesis may not contribute equally to the mutation rate of HIV-1. The data also provide estimates of substitution and frameshift error rates during transcription by T7 RNA polymerase.


Elevated expression of monocyte chemoattractant protein 1 by vascular smooth muscle cells in hypercholesterolemic primates. Atherosclerosis is marked by an overt inflammatory infiltrate, with enhanced recruitment of monocytes/macrophages observed in both human and experimental atherosclerosis. We previously determined that monocyte chemoattractant protein 1 (MCP-1) accounts for virtually all of the chemotactic activity produced by vascular (aortic) smooth muscle cells in culture. We now report that arteries from a primate model of atherosclerosis with dietary-induced hypercholesterolemia exhibit increased levels of MCP-1 mRNA expression in vivo, whereas their normal counterparts demonstrate minimal MCP-1 expression. Furthermore, immunohistochemistry and in situ hybridization clearly indicate that the expression of MCP-1 protein and mRNA is in the smooth muscle cells of the medial layer of the artery and in monocyte-like and smooth muscle-like cells found in the overlying intimal lesion. These studies indicate that one of the responses to dietary hypercholesterolemia is the expression of MCP-1 by vascular smooth muscle cells. This expression, when augmented with other cellular and molecular factors, could significantly contribute to the recruitment of monocytes/macrophages to the vessel wall.


A high-molecular-weight squid neurofilament protein contains a lamin-like rod domain and a tail domain with Lys-Ser-Pro repeats. Previous studies have shown that two low molecular-weight neurofilament (NF) proteins (NF-60 and NF-70) from the squid Loligo pealei are translated from mRNAs that are splice variants of a single squid NF gene. In this study, we report the isolation and characterization of cDNA clones encoding a high-molecular-weight squid NF protein (NF-220), the mRNA of which derives from the same squid NF gene. All three proteins are identical in their amino-terminal and lamin-like rod domains but differ in their carboxyl-terminal tail regions. In contrast to the short tail domains of NF-60 and NF-70, the NF-220 protein has a longer tail domain containing an acidic cluster of amino acids immediately followed by repeated copies of the sequence motif Lys-Ser-Pro. The Lys-Ser-Pro domain is similar to that of mammalian medium NF (NF-M) and high NF (NF-H) proteins, where the serines are highly phosphorylated. Except for these Lys-Ser-Pro motifs, there is surprisingly little structural similarity between the squid NF-220 protein and mammalian NF-M and NF-H proteins. Furthermore, the location of introns in squid NF-220 protein shows that it is more closely related to nuclear lamins and type III intermediate-filament proteins than to vertebrate NF proteins.


RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase. High-affinity ligands of the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) were isolated by the SELEX procedure (systematic evolution of ligands by exponential enrichment) from RNA populations randomized at 32 positions. Analysis of these ligands revealed a pseudoknot consensus with primary sequence bias at some positions. We demonstrated that at least one of the ligands inhibits cDNA synthesis by HIV reverse transcriptase but fails to inhibit other reverse transcriptases. These experiments highlight the power of SELEX to yield highly specific ligands that reduce the activity of target proteins. Such ligands may provide therapeutic reagents for viral and other diseases.


Identification of a mammalian gene structurally and functionally related to the CDC25 gene of Saccharomyces cerevisiae. The yeast Saccharomyces cerevisiae CDC25 gene encodes a nucleotide-exchange-factor (NEF) that can convert the inactive GDP-bound state of RAS proteins to an active RAS-GTP complex. CDC25 can activate the yeast RAS proteins as well as the human H-ras protein. CDC25 is a member of a family of yeast genes that likely encode NEFs capable of regulating the RAS-related proteins found in yeast. By aligning the amino acid sequence of CDC25-related gene products we found a number of conserved motifs. Using degenerate oligonucleotides that encode these conserved sequences, we have used polymerase chain reactions to amplify fragments of mouse and human cDNAs related to the yeast CDC25 gene. We show that a chimeric molecule, part mouse and part yeast CDC25, can suppress the loss of CDC25 function in the yeast S. cerevisiae.


Design of peptide-acridine mimics of ribonuclease activity. A series of peptide-acridine conjugates was designed and synthesized, based on three features of the proposed catalytic mechanism of RNase A: 2'-proton abstraction by His-12, proton donation to the leaving 5'-oxygen by His-119, and stabilization of the pentacoordinated phosphorous transition state by Lys-41. The substrate binding capability of RNase A was mimicked by the intercalator, acridine. Lysine served as a linker between acridine and the catalytic tripeptide. Cleavage of target RNA was monitored by agarose gel electrophoresis and by gel-permeation chromatography. The carboxyl-amidated conjugates HGHK(Acr)-NH2, HPHK(Acr)-NH2, and GGHK(Acr)-NH2 (where Acr indicates 2-methyl-9-acridinemethylene) all had similar hydrolytic activity. The catalytic mechanism most likely involved only the abstraction of the 2'-proton and stabilization of the transition state. These RNase mimics utilized rRNA and single-stranded RNA but not double-stranded RNA and tRNA as substrates.


Central role for differential gene expression in mammalian hibernation. Mammalian hibernators experience dramatic reductions in body temperature, metabolic rate, respiratory rate, and heart rate during hibernation. These changes are precisely controlled and reversible with only internally driven mechanisms, suggesting specific biochemical regulation. We present a model that integrates our observations of differential liver gene expression during preparation for, and maintenance of, the hibernating state, with the known phylogenetic interspersion of hibernating species in several major mammalian lineages. This model predicts a major role for the differential expression of existing mammalian genes in the biochemical regulation of hibernation.


copia-like retrotransposons are ubiquitous among plants. Transposable genetic elements are assumed to be a feature of all eukaryotic genomes. Their identification, however, has largely been haphazard, limited principally to organisms subjected to molecular or genetic scrutiny. We assessed the phylogenetic distribution of copia-like retrotransposons, a class of transposable element that proliferates by reverse transcription, using a polymerase chain reaction assay designed to detect copia-like element reverse transcriptase sequences. copia-like retrotransposons were identified in 64 plant species as well as the photosynthetic protist Volvox carteri. The plant species included representatives from 9 of 10 plant divisions, including bryophytes, lycopods, ferns, gymnosperms, and angiosperms. DNA sequence analysis of 29 cloned PCR products and of a maize retrotransposon cDNA confirmed the identity of these sequences as copia-like reverse transcriptase sequences, thereby demonstrating that this class of retrotransposons is a ubiquitous component of plant genomes.


Levels of mRNA coding for motoneuron growth-promoting factors are increased in denervated muscle. Partial denervation of skeletal muscle induces sprouting of axons remaining within the muscle, possibly as a result of increased synthesis by denervated muscle fibers of motoneuron growth-promoting factors. Direct verification of this hypothesis has not been possible because the molecules responsible are not unambiguously characterized. We used Xenopus oocytes as a functional assay for mRNAs coding for secreted growth factors: preparations of mRNA from innervated and denervated neonatal muscle were injected into oocytes. Three days later, oocytes injected with denervated muscle mRNA expressed increased levels of nicotinic acetylcholine receptor and voltage-dependent sodium channels at their membrane. Proteins secreted by the same oocytes were tested for their effects on (i) neurite outgrowth from embryonic chicken ventral spinal cord neurons; (ii) survival in mixed culture of embryonic chicken motoneurons identified using the SC1 antibody; and (iii) survival of embryonic motoneurons purified by panning on SC1 antibody. In all three assays, media conditioned by oocytes injected with mRNA from denervated muscle contained significantly higher levels of biological activity than did those from oocytes injected with innervated muscle mRNA or water. mRNA was prepared from muscle at different times after denervation: a maximal increase was obtained already after 1 day, consistent with an involvement in sprouting. Synthesis of motoneuron growth-promoting factors is thus regulated by denervation in a parallel fashion to that of other key components of the neuromuscular junction.


Role of p34cdc2-mediated phosphorylations in two-step activation of pp60c-src during mitosis. Phosphorylation of pp60c-src by p34cdc2 at three amino-proximal serine/threonine residues is temporally correlated with, but insufficient for, mitotic activation of c-Src kinase. The direct cause of activation during mitosis appears to be temporally correlated partial dephosphorylation of Tyr-527, a residue whose phosphorylation strongly suppresses pp60c-src activity. Site-directed mutagenesis of the serine/threonine phosphorylation sites blocks half the mitosis-specific decrease in Tyr-527 phosphorylation and half the increase in pp60c-src kinase activity. We conclude that p34cdc2 partially activates pp60c-src by a two-step process in which its serine/threonine phosphorylations either sensitize pp60c-src to a Tyr-527 phosphatase or desensitize it to a Tyr-527 kinase. Furthermore, additional events, independent of these p34cdc2-mediated phosphorylations, participate in mitotic activation of pp60c-src.


The glial voltage-gated sodium channel: cell- and tissue-specific mRNA expression. Previous electrophysiological and pharmacological studies on central and peripheral glia revealed the presence of voltage-gated Na channels with properties that are similar but not identical to those of neuronal Na channels. Here we report the isolation and characterization of a cDNA encoding the C-terminal portion of a putative glial Na-channel (Na-G) alpha subunit. The amino acid sequence deduced from this cDNA indicates that the Na-G represents a separate molecular class within the mammalian Na-channel multigene family. By Northern blot, RNase protection, and in situ hybridization assays, we demonstrate that, in addition to brain astroglia, the Na-G mRNA is expressed in cultures of Schwann cells derived from dorsal root ganglia or from sciatic nerve. In vivo, the Na-G mRNA is detected not only in brain, dorsal root ganglia, and sciatic nerve, but also in tissues outside the nervous system including cardiac and skeletal muscle and lung. Its level varies according to the tissue and is developmentally regulated. The sequence and expression data concur in designating Na-G as an distinct type of Na channel, presumably with low sensitivity to tetrodotoxin.


[Atypical verbal behavior in a 30 year follow-up of an acquired aphasia-epilepsy syndrome]. Detailed study of the communication of one of the patients followed-up by Deonna et al. (1989) who has been suffering since age seven of an atypical acquired aphasia-epilepsia syndrome. Seizures as well as peculiar verbal productions persist at adult age. After a thirty year follow-up, only a small lexicon has been acquired, mostly made of onomatopeia and idiosyncrasies. We describe this lexicon and discuss the quality of the communication in a real-life ecological situation. We then compare it to the jargon and the aphasic productions of the standard neuropsychological examination.


Early manifestations of dementing illness: treatment with glycosaminoglycan polysulfate. 1. In a multicenter, placebo-controlled, double-blind clinical trial in 156 elderly patients with psychopathologic symptoms, glycosaminoglycan polysulfate was found to be a therapeutically effective agent in the treatment of the earliest manifestations of a dementing process. 2. Treatment with glycosaminoglycan polysulfate in the daily dosage of 600 LRU, administered on the basis of a divided dosage schedule for 24 weeks, was significantly superior to an inactive placebo on several outcome measures including the SCAG Total and factor scores (i.e., Cognitive Dysfunction, Withdrawal, Agitation/Irritability and Depression), the NOWLIS Total and Fatiguability factor scores, the MMSE, the HAM-D Total and Vegetative Symptoms factor score and the CGI Severity of Illness and Global Improvement. 3. The drug was well tolerated; vital signs and laboratory measures did not show clinically significant changes within the experimental period.


Bleomycin/cis-platin as neoadjuvant chemotherapy before radical radiotherapy in localized, inoperable carcinoma of the esophagus. A prospective randomized multicentre study: the second Scandinavian trial in esophageal cancer. Survival and swallowing function were studied in a randomized trial of 97 patients with inoperable, localized esophageal carcinoma. Radical radiotherapy was given to 51 patients, while 46 patients had two courses of bleomycin/cisplatin before radiotherapy. The survival was 29% after one year, and 6% after 3 years in the radiotherapy group. The survival in the combined treatment group was 18 and 0%, respectively; p = 0.1895. The number of patients who could swallow any food increased from 6% before treatment to 38% after 3 months in the radiotherapy group, and from 0% to 23% in the combined group. No benefit was found by combining bleomycin/cisplatin with radiotherapy.


[Stomach carcinoma: surgical strategy and therapy results 1992]. Surgical strategy for gastric carcinoma consists in total gastrectomy as a rule combined with extended lymphadenectomy. In small tumors of the intestinal type located in the distal stomach distal gastric resection can be taken into consideration. In case of high risk patients endoscopic polypectomy of a polypoid early gastric carcinoma can be sufficient. If the operation is done for cure we perform intraoperative radiotherapy within a prospective trial. For palliative situations procedures including removal of the tumor have better results with regard to quality of life and survival than bypass methods.


Chloride-dependent cation conductance activated during cellular shrinkage. A chloride (Cl-)-dependent, nonselective cation conductance was activated during cellular shrinkage and inhibited during cellular swelling or by extracellular gadolinium. The shrinking-induced, nonselective cation conductance and the swelling-induced anion conductance appear to function in the regulation of cell volume in airway epithelia. The shrinking-induced cation conductance had an unusual dependence on Cl-: partial replacement of extracellular Cl- with aspartate reduced the magnitude of the shrinking-enhanced current without accompanying changes in the reversal potential. The Cl- dependence of the nonselective cation conductance could provide a mechanism that tightly regulates Cl- secretion and sodium reabsorption in cells under osmotic stress.


Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes. The DNA sequence of 91.4 kilobases of the Escherichia coli K-12 genome, spanning the region between rrnC at 84.5 minutes and rrnA at 86.5 minutes on the genetic map (85 to 87 percent on the physical map), is described. Analysis of this sequence identified 82 potential coding regions (open reading frames) covering 84 percent of the sequenced interval. The arrangement of these open reading frames, together with the consensus promoter sequences and terminator-like sequences found by computer searches, made it possible to assign them to proposed transcriptional units. More than half the open reading frames correlated with known genes or functions suggested by similarity to other sequences. Those remaining encode still unidentified proteins. The sequenced region also contains several RNA genes and two types of repeated sequence elements were found. Intergenic regions include three "gray holes," 0.6 to 0.8 kilobases, with no recognizable functions.


The skeletal muscle chloride channel in dominant and recessive human myotonia. Autosomal recessive generalized myotonia (Becker's disease) (GM) and autosomal dominant myotonia congenita (Thomsen's disease) (MC) are characterized by skeletal muscle stiffness that is a result of muscle membrane hyperexcitability. For both diseases, alterations in muscle chloride or sodium currents or both have been observed. A complementary DNA for a human skeletal muscle chloride channel (CLC-1) was cloned, physically localized on chromosome 7, and linked to the T cell receptor beta (TCRB) locus. Tight linkage of these two loci to GM and MC was found in German families. An unusual restriction site in the CLC-1 locus in two GM families identified a mutation associated with that disease, a phenylalanine-to-cysteine substitution in putative transmembrane domain D8. This suggests that different mutations in CLC-1 may cause dominant or recessive myotonia.


Identification of a protein that binds to the SH3 region of Abl and is similar to Bcr and GAP-rho. A Src homology 3 (SH3) region is a sequence of approximately 50 amino acids found in many nonreceptor tyrosine kinases and other proteins. Deletion of the SH3 region from the protein encoded by the c-abl proto-oncogene activates the protein's transforming capacity, thereby suggesting the participation of the SH3 region in the negative regulation of transformation. A complementary DNA was isolated that encoded a protein, 3BP-1, to which the SH3 region of Abl bound with high specificity and to which SH3 regions from other proteins bound differentially. The sequence of the 3BP-1 protein is similar to that of a COOH-terminal segment of Bcr and to guanosine triphosphatase-activating protein (GAP)-rho, which suggests that it might have GAP activity for Ras-related proteins. The 3BP-1 protein may therefore be a mediator of SH3 function in transformation inhibition and may link tyrosine kinases to Ras-related proteins.


From DSM-I to III-R; voices of self, mastery and the other: a cultural constructivist reading of U.S. psychiatric classification. The continual process of mental disease classification in U.S. psychiatry is assumed to reflect advancing professional knowledge of these disorders. To date, the American Psychiatric Association has developed four standard classifications, or nosologies, called Diagnostic and Statistical Manuals ('DSMs'). DSM-I, the earliest, appeared in 1952 while the most recent, DSM-III-R, appeared in 1987. This paper employs a cultural constructivist perspective to deconstruct these nosologies and the classificatory process itself. Constructivism's premises, which emphasize culture, history, meaning and the constructed nature of medical phenomena, serve as the framework for the analysis. The paper shows that professional psychiatric classification expresses an underlying cultural psychology which encompasses four phenomenological domains and one of three Western person conceptions. Classifications are found to be explorations of culturally meaningful etiologies that explain the absence of 'self control', a central ethnopsychological aspect of the idealized self. Consideration of the vantage point of the voice of classification indicates that the ideal self is gender- (male), ethnic- (German Protestant) and age-specific (adult). The ethnic self's essence, and that of the Other, is believed to be biological, itself assumed to be natural and beyond culture or bias. Consequently, the ethnopsychology constructs as biologically caused the real and imagined differences in the gender, age or culturally Other. This invidious ethnobiological essentialism acts to create and maintain self-worth through a radical differentiation of self from those represented as Other.


Prostate specific antigen levels during and after external beam radiotherapy for localized carcinoma of the prostate: predictor of therapeutic efficacy. For 105 patients with locoregional carcinoma of the prostate, prostate specific antigen (PSA) levels were evaluated before, during and after external beam radiotherapy. The median follow-up is 17 months. In 51 patients (48.5%) initial PSA levels exceeded the maximum normal value of 20 ng/ml. Nine patients kept non-declining high levels just after radiotherapy. Only one of these is free of disease. Assuming PSA levels decrease exponentially during radiotherapy, a mean half-life of 62 days (median 54, SD 26 days) was calculated. Three out of five patients with a PSA half-life of more than 88 days relapsed as compared to a 8% (3/37) relapse rate in patients with a "normal" half-life. Prolonged PSA half-life suggests residual disease. PSA levels are expected to further decrease after radiation. Six months after irradiation persistent high PSA levels were found in 14/51 (27.5%) patients. Only four of them had no evidence of manifest disease. Important negative prognostic factors for disease control in our series were non-declining high levels of PSA, a PSA serum half-life exceeding 88 days and persistence of elevated PSA values longer than six months after treatment. In our opinion, PSA is a valuable marker in the follow-up of prostate cancer patients during and after radiotherapy.


[Rapeseed poisoning of wild herbivores]. Beginning with the simultaneous occurrence of the first extensive sowing of 00-rape and local increased losses among hares and roe deer in Western Germany and Austria at the end of 1986, the clinical and morphological symptoms of rape poisoning are discussed. They consist of damage to endo- and epithelium, cell membranes, blood, liver and in the so called "rape-blindness". Subsequently, the most important toxic agents of rape including their metabolites are presented. They consist in alkenyl- and indolyl-glucosinolates, leading to isothiocyanates (mustard oils), thiocyanates or thiocyanate ions resp., nitriles and antithyroid agents (e.g. goitrin) as well as S-methylcysteine sulphoxide and its metabolites, particularly dimethyl disulphide. Finally, the activity spectrum of the toxic agents or the metabolites and the clinical picture of the affected wildlife in 1986 are compared with the result that the losses of that period are most likely to be traced back to rape poisoning and that the rape-blindness mentioned is to be interpreted as a thiocyanate-psychosis.


Effect of mismatching serologically defined residues on kidney transplant survival. Amino acids within the HLA class I and II molecules were first examined individually for correlation with antibody reactions of over 50,000 antisera. The amino acids that correlated with the serologic reactions were classified as serologically defined: approximately half of the amino acids fell within that category, and the other half were employed as controls. The cadaver donor kidney transplant survival for patients who were mismatched for each of the sera-defined amino acids were compared to mismatches of the nonserologically defined amino acids. No differences were noted in the graft survivals of patients with mismatches for the serologically defined amino acids of HLA-A, -B, and -DR loci and for the nonserologically defined amino acids. Since many of the patients also had additional amino acids of mismatch, we cannot yet conclude that the serologically defined amino acids do not significantly influence graft outcome. In other words, the test may not have been sensitive enough to detect the degree of immunogenicity that may exist. However, we can conclude that mismatches for no single amino acid are strongly immunogenic.


Prevalence of hemoglobinopathies in north Jordan. Blood samples from 1,000 subjects, 2-80 years old, were tested to explore the prevalence of alpha-thalassemia trait, high persistent HbF (HPFH), sickle cell trait and beta-thalassemia minor in northern Jordan. Hematological parameters and hemoglobin electrophoresis were carried out on all samples. Results showed 10 (1%) subjects were sickle cell trait, 35 (3.5%) were heterozygous beta thalassemia, 31 (3.1%) alpha thalassemia trait and 10 (1%) high persistent HbF. The prevalence rates were different from those reported in neighbouring countries and their significance is discussed. No other abnormal hemoglobin types were observed.


Purification of larval Taenia solium antigens by gel filtration. Crude larval Taenia solium extracts were fractionated by Sephacryl S-200 gel filtration into four fractions (W1-W4). The sensitivities of the fractions to rabbit and pig antiserum against Taenia solium were tested by double immunodiffusion, immunoelectrophoresis, and ELISA. Fraction W2 which was highly sensitive to antisera was shown by immunoblotting to contain antigen B (95 and 105 kDa). The four fractions were shown to contain antigenic determinants common with pig serum proteins and crude extracts of other Platyhelminthes (especially Taenia hydatigena). Fraction W2 has the potential to be used as a serodiagnostic antigen.


Variation amongst the neutralizing epitopes of bluetongue viruses isolated in the United States in 1979-1981. Neutralizing epitopes present on field isolates of bluetongue virus (BTV) serotypes 10, 11, 13 and 17 were evaluated with a panel of polyclonal and neutralizing monoclonal antibodies (MAbs). A total of 91 field isolates were evaluated, including 15 isolates of BTV-10, 29 isolates of BTV-11, 26 isolates of BTV-13, and 21 isolates of BTV-17. The viruses were isolated from cattle, goats, sheep, elk and deer in Idaho, Louisiana, Nebraska and, predominantly, California, in the years 1979, 1980 and 1981. The isolates were analyzed and compared using a panel of neutralizing MAbs which included five MAbs raised against BTV-2, seven against BTV-10, five against BTV-13, and six against BTV-17. Neutralization patterns obtained with the MAb panel and individual field isolates were compared to those obtained with prototype viruses of each serotype. All field isolates were neutralized by at least some of the MAbs raised against the prototype virus of the same serotype. All field isolates of BTV-10 were neutralized by the seven MAbs raised to BTV-10, whereas the field isolates of BTV-11, BTV-13 and BTV-17 were not consistently neutralized by all of the MAbs raised against the prototype virus of the same serotype. Variation in neutralizing epitopes recognized by the MAb panel was most pronounced amongst the field isolates of BTV-17. A one-way cross neutralization was evident between BTV-10 and BTV-17 as all field isolates of BTV-17 were neutralized by four of the MAbs raised against BTV-10. In contrast, no BTV-10 isolates were neutralized by the MAbs raised against BTV-17. Differences in the MAb neutralization patterns of field isolates of BTV-11, BTV-13 and BTV-17 suggest that the immunogenic domain responsible for their neutralization is plastic, such that individual epitopes within the domain may vary in their significance to the neutralization of different viruses, even of the same serotype. The apparent conservation of neutralizing epitopes on field isolates of BTV-10 suggests that the field isolates may be derived from the modified-live vaccine strain of BTV-10.


Polymorphonuclear neutrophil leukocyte function in clinical bovine patients and in cows with or without Staphylococcus aureus mastitis. A fluorochrome microassay was used to investigate peripheral blood polymorphonuclear leukocyte (PMNL) function in cattle. Glass-adherent PMNL were reacted with Staphylococcus aureus preincubated in 20% bovine serum for 30, 60 and 90 min. Coverslips were stained with acridine orange (AO) followed by crystal violet to quench extracellular bacterial fluorescence. PMNL function was evaluated by counting the number of dead (stained red with AO) and live (stained green with AO) S. aureus contained within 100 PMNL. A phagocytic index was calculated as the average number of bacteria contained within PMNL. The percentage killing of S. aureus was calculated from the average proportion of S. aureus within PMNL that were dead. Six clinically normal Holstein calves, 3-4 months of age, were sampled on 6 consecutive days. PMNL phagocytosis and killing did not vary significantly (p greater than 0.05) among repeated samplings per calf. PMNL function increased with increasing time of incubation of PMNL with S. aureus. Means (+/- SD) for percentage killing were 46.7 +/- 13.1, 57.4 +/- 11.6, and 62.1 +/- 9.8% for 30, 60 and 90 min of reaction, respectively. Means (+/- SD) for the phagocytic index were 2.9 +/- 0.8, 3.6 +/- 1.0, and 4.2 +/- 1.1 bacteria/PMNL for 30, 60 and 90 min of reaction, respectively. PMNL function was determined in 30 normal cattle of various breeds, age and sex, and these values were pooled to provide normal values for PMNL function. When values for bovine clinical patients (n = 25) with various diagnoses were compared with normal values (defined by the mean +/- 2SD for the 30 normal cattle) for PMNL function, only one patient was observed to exhibit PMNL hypofunction. A cow with disseminated intravascular coagulation in association with peracute coliform mastitis exhibited decreased PMNL killing capacity. Abnormal PMNL function was uncommon in the hospital population studied. Peripheral blood PMNL function was evaluated in lactating Holstein cows with (n = 15) or without (n = 15) chronic subclinical S. aureus mastitis. There was no significant (p greater than 0.05) difference in PMNL function among these cows.


[An assessment of the status of natural foci of Crimean-Congo hemorrhagic fever in the Crimea]. The parasitological data and the results of the virological and serological investigations of materials, collected in nature and in the course of study of the immune structure of the population, are indicative of the circulation of CHF virus in the Crimea and the possibility of human infection. Data on spontaneous infection of four species of Ixodes ticks with CHF virus have been confirmed, including the data, obtained for the first time for this region, on the participation of Dermacentor marginatus in this process. The study has revealed, also for the first time, that, together with European brown hares, the natural foci of this infection may be maintained by scilly shrews, common voles and European wood mice. Low activity of the Crimean focus may be the result of active land reclamation, keeping the cattle stalled in most of the stock-breeding farms of the region, as well as a sharp decline in the number of hares at the territories of hunting preserves.


[The improvement of the immunodiagnosis of para-influenza infection based on erythrocyte reagents]. Study of different methods of hemosensitization helped detect the optimum technology for the preparation of parainfluenza (type 3) antigenic erythrocyte diagnosticum. This technology ensures the highest sensitivity, specificity, economy (with respect to viral antigen consumption) and stability of the reagent. Titration of antibodies by means of the new diagnosticum and the detection of parainfluenza cases among children with acute respiratory virus infections, acute and chronic glomerulonephritis have been highly effective, considerably more effective than similar determinations by the hemagglutination inhibition test.


[The capacity of purified staphylococcal anatoxin to correct antigen-specific and antigen-nonspecific immunological defects]. Purified staphylococcal toxoid is capable of partially preventing the development of antigen-specific (induced by the supraoptimal dose of sheep red blood cells) and antigen-nonspecific (induced by Tahyna virus) defects of humoral immune response, as well as abolishing these defects. The presence and manifestation of the correction of virus-induced immunodeficiency is determined by the dose of the toxoid and the interval between the injections of purified staphylococcal toxoid and the infective agent.


[Stomach cancer in pregnancy].

[Stomach cancer in pregnancy]. The presented clinical course of a 40 year old Primipara, in whom gastric cancer during pregnancy occurred, demonstrates the difficulties of differential diagnosis in early stages of the disease because of the non specific symptoms at that time. Mostly, they are related to physiological pregnancy associated complaints. Therefore, diagnosis is very often obtained lately and connected with advanced clinical stages of the tumor, which causes the bad survival rates of 10-20% of these young women.


Molecular definition of the smallest region of deletion overlap in the Wolf-Hirschhorn syndrome. Wolf-Hirschhorn syndrome (WHS), associated with a deletion of chromosome 4p, is characterized by mental and growth retardation and typical facial dysmorphism. A girl with clinical features of WHS was found to carry a subtle deletion of chromosome 4p. Initially suggested by high-resolution chromosome analysis, her deletion was confirmed by fluorescence in situ hybridization (FISH) with cosmid probes, E13 and Y2, of D4S113. To delineate this 4p deletion, we performed a series of FISH and pulsed-field gel electrophoresis analyses by using probes from 4p16.3. A deletion of approximately 2.5 Mb with the breakpoint at approximately 80 kb distal to D4S43 was defined in this patient and appears to be the smallest WHS deletion so far identified. To further refine the WHS critical region, we have studied three unrelated patients with presumptive 4p deletions, two resulting from unbalanced segregations of parental chromosomal translocations and one resulting from an apparently de novo unbalanced translocation. Larger deletions were identified in two patients with WHS. One patient who did not clinically present with WHS had a smaller deletion that thus eliminates the distal 100-300 kb from the telomere as being part of the WHS region. This study has localized the WHS region to approximately 2 Mb between D4S43 and D4S142.


[The dynamics of latent inhibition in rats under the action of substance P]. The role of substance P in latent inhibition was studied in experiments on rats. Administration of neuropeptide during pre-exposition of conditioned stimulus and before conditioning disturbed all signs of latent inhibition: level of reproduction, retention and resistance to amnestic action of conditioned reaction in the task of passive avoidance. Single administration of haloperidol before learning prevented the disturbance. Significance of hyperfunction of substance P in selective attention and pathogenesis of schizophrenia is discussed.


[Prenatal serum screening for Down's syndrome]. With the aid of two commercially available analysis programmes we effected non-invasive assessment of the risk for Down's syndrome in 597 pregnancies with healthy fetuses and in 22 pregnancies with trisomie 21-affected fetuses, maternal age ranging between 18 and 45 years. Based on the women's serum levels, hormone concentrations of AFP, free estriol and beta HCG were determined, a multiple of their median was combined with the age factor, and the result used as a parameter for overall risk estimation. A risk cut-off-level greater then 1:250 was considered to represent a positive risk. For patients younger than 35 years test sensitivity, at just over 50%, was found to be low. For those aged 35 years or greater, however, the Dermalog programme was able to detect 94.4% and the Alpha programme 88.2% of fetuses affected by trisomy 21, with a false-positive rate of 19.6 and 19.1%, respectively. If the test were to be applied to all pregnant women beyond 35 years of age the detection rate of Down's syndrome could be nearly doubled compared to today's figure (only 50%, of women in this age group undergo amniocentesis). By the same token the rate of amniocentesis-induced abortion (approx. 1%) could be reduced to about one third.


Congenital deficiency of alpha-fetoprotein. Although alpha-fetoprotein may play a role in fetal immune function or in maintenance of osmotic pressure, its exact function is unknown. We report two infants documented to have congenital deficiency of alpha-fetoprotein. One infant had cord blood levels less than 0.5 ng/ml. The second infant had a neonatal level of 120 ng/ml, which is about 2% of the usual concentration for a term newborn. These infants document the existence of congenital deficiency of serum alpha-fetoprotein. Because it is homologous to albumin, congenital deficiency of alpha-fetoprotein may be analogous to analbuminemia, a benign genetic trait.


"Doing battle": a metaphorical analysis of diabetes mellitus among Navajo people. Effective communication with patients and their family members forms the foundation of a therapeutic relationship. This is particularly important when the occupational therapist, other health professionals, and the patient are from different cultural backgrounds. This paper describes one aspect of the findings of a ethnographic study of chronic diabetes among the Navajo people (referred to here as Dine'). It focuses on the dominant metaphorical images that were used by the informants to describe their illness experiences. The data suggest that diabetes can be considered a metaphor for larger social changes in the life-style and traditions (e.g., away from sheepherding as a means of basic subsistence to obtaining urban-centered employment) of native Americans and their effects on the Dine'. Implications of our findings include the importance of metaphorical communication for perceptions of compliance, powerlessness, and patient and therapist satisfaction with the therapeutic relationship.


Hmong children and their families: consideration of cultural influences in assessment. Occupational therapists assessing young Hmong children with developmental problems must consider their families' cultural beliefs as they affect the design of assessment procedures and practices. Choices that families make about health and educational services are influenced by their beliefs. Developmental status can be affected by unresolved medical problems and the child's general health condition. Assessment components based on cultural awareness may improve the effectiveness of early identification of Hmong children with developmental delay. Appropriate use of interpreters, creation of the most beneficial assessment environment, parental report, and observation of functional skills and play provide needed information when determining the child's eligibility for early intervention services. The author has found that trained interpreters provide the most reliable communication between family members and the therapist. Assessments in the home environment are encouraged due to the child's age and the need for family support and interaction. Parents are an excellent source of information about the child's current and past functional abilities. Observations of the child's interaction with family members, with objects and toys during play, and during functional daily living activities provides the therapist with valuable information about the child's need for intervention.


Immune electron microscopic characterization of monoclonal antibodies to Alzheimer neurofibrillary tangles. Characterization of eleven monoclonal antibodies (MAbs), raised to isolated sodium dodecyl sulfate (SDS)-treated Alzheimer's neurofibrillary tangles (ANT), has revealed the presence of at least two different epitopes. MAbs were tested for reactivity to ubiquitin and paired helical filaments (PHF) isolated by three different procedures. The effect of protease and/or alkaline phosphatase pretreatment on the reactivity of the MAbs with isolated PHF was also examined. All MAbs that had reacted strongly in the ELISA with sonicated SDS-treated ANT also immune decorated isolated PHF to varying degrees. Two MAbs exhibited a high reactivity to PHF: 3-39 and 5-25. MAb 3-39 was found to recognize a protease sensitive epitope. In contrast MAb 5-25 was found to consistently decorate isolated PHF in all preparations and exhibited a strong reactivity to ubiquitin, and the epitope in isolated PHF was not protease sensitive. Thus structural PHF after protease treatment and detergent treatment contain an antigenic site that is present in ubiquitin.


Expression of tenascin by vascular smooth muscle cells. Alterations in hypertensive rats and stimulation by angiotensin II. The extracellular matrix glycoprotein tenascin is associated with remodeling events in many embryonic and pathologic tissues. The expression of tenascin has been investigated by immunohistochemistry in blood vessels of Wistar-Kyoto (normotensive) and spontaneously hypertensive rats. Weak tenascin staining was present throughout the tunica media of large and small arteries from normotensive animals; strong staining was only detectable at branching sites. In arteries from hypertensive animals, foci of strong tenascin staining were scattered throughout the tunica media. The expression of tenascin mRNA and protein by rat aortic smooth muscle cells cultured in serum-free medium was induced by the vasoconstrictor peptide angiotensin II. Transforming growth factor-beta and platelet-derived growth factor also stimulated tenascin mRNA expression. Vascular smooth muscle cells attached specifically to a substratum of tenascin, but remained rounded. Thus, increased focal tenascin expression by vascular smooth muscle cells is associated with hypertension, and may mediate angiotensin II-induced changes in vascular structure in hypertension.


Differential expression of proteoglycans on the surface of human melanoma cells characterized by altered experimental metastatic potential. Heparan sulphate (HS) and chondroitin sulphate (CS) proteoglycans (PGs) frequently have opposite biologic functions in cell-matrix adhesion as well as in the regulation of cell proliferation. Data revealed that sulphated glycosaminoglycans (sGAGs) (sugar chains of PGs) are differently expressed in tumor cells characterized by different metastatic potential; the more metastatic cells contain a higher HS/CS ratio. As the proliferative capacity of tumor cells is also frequently altered in parallel with their metastatic potential, it was not clear whether observed PG alterations reflect changes in cell proliferation or metastatic potential. The cell-associated PG expression and sGAG biosynthesis was studied in tumor cells of human melanoma lines characterized by different experimental metastatic potential to the mouse liver but similar in vitro/in vivo proliferation rates. Using antibodies against PGs we found different expression of PG epitopes in melanoma lines, except from the melanoma antigen. Unlike the low CSPG (melCSPG) metastatic melanoma cells, the cell line with high metastatic capacity contained a higher proportion of positive cells for surface-HSPG without the coexpression of certain cartilage-type CSPG epitopes (recognized by MAb HSFPG 529) as well as by an increased pericellular HS/CS ratio due to intracellular accumulation/retention of CS. Immunocytochemistry of adherent cells revealed HSPGs at substrate-attached membrane areas only in cases of highly metastatic melanoma cells. These data further support our view that the absolute or relative dominance of HSPGs over CSPGs at the cell surface of metastatic tumor cells can be considered a marker of a more metastatic phenotype.


Keratin expression in cervical cancer.

Keratin expression in cervical cancer. Using a panel of 21 monoclonal and 2 polyclonal keratin antibodies, capable of detecting separately 11 subtypes of their epithelial intermediate filament proteins at the single cell level, we investigated keratin expression in 16 squamous cell carcinomas, 9 adenocarcinomas, and 3 adenosquamous carcinomas of the human uterine cervix. The keratin phenotype of the keratinizing squamous cell carcinoma was found to be most complex comprising keratins 4, 5, 6, 8, 13, 14, 16, 17, 18, 19, and usually keratin 10. The nonkeratinizing variety of the squamous cell carcinoma expressed keratins 6, 14, 17, and 19 in all cases, usually 4, 5, 7, 8, and 18, and sometimes keratins 10, 13, and 16. Adenocarcinomas displayed a less complex keratin expression pattern comprising keratins 7, 8, 17, 18, and 19, while keratin 14 was often present and keratins 4, 5, 10 and 13 were sporadically found in individual cells in a few cases. These keratin phenotypes may be useful in differential diagnostic considerations when distinguishing between keratinizing and nonkeratinizing carcinomas (using keratin 10, 13, and 16 antibodies), and also in the distinction between nonkeratinizing carcinomas and poorly differentiated adenocarcinomas, which do not express keratins 5 and 6. Keratin 17 may also be useful in distinguishing carcinomas of the cervix from those of the colon and also from mesotheliomas. Furthermore the presence of keratin 17 in a CIN I, II, or III lesion may indicate progressive potential while its absence could be indicative of a regressive behavior. Because most carcinomas express keratins 8, 14, 17, 18, and 19, we propose that this expression pattern reflects the origin of cervical cancer from a common progenitor cell, i.e., the endocervical reserve cell that has been shown to express keratins 5, 8, 14, 17, 18, and 19.


Antigenic variation among transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus strains detected with monoclonal antibodies to the S protein of TGEV. Five nonneutralizing monoclonal antibodies (MAb) generated to the virulent Miller strain of transmissible gastroenteritis virus (TGEV) and specific for the S protein were characterized. Competition assays between purified and biotinylated MAb indicated that MAb 75B10 and 8G11 mapped near a new subsite, designated V and 2 MAb, 44C11 and 45A8, mapped to a previously designated subsite D. A fifth MAb mapped between subsites V and E. These MAb were tested with 3 previously characterized MAb to subsites A, E, and F in fixed-cell ELISA and cell culture immunofluorescent assays against 5 reference and 9 field strains of TGEV and 2 US strains (ISU-1 and ISU-3) of porcine respiratory coronavirus (PRCV). Subsites A, E, and F were conserved on all TGEV and PRCV strains examined. The 2 MAb to subsite V, 8G11 and 75B10, reacted only with the Miller TGEV strains (M5C, M6, and M60), except that 75B10 also recognized field strain U328. The MAb 11H8 did not react with 4 field strains or the Purdue strains of TGEV. The 2 MAb to subsite D reacted with all TGEV strains examined, but not with 2 US PRCV strains, 2 European PRCV strains, 1 feline infectious peritonitis virus strain, and 1 canine coronavirus strain. Because of this specificity for TGEV, but not PRCV, these latter 2 subsite D MAb may be useful for the development of competition ELISA to differentiate serologically between TGEV and PRCV infections in swine, similar to the currently used European subsite D MAb.


Biochemical study on the process of skin graft take. Investigations on the process of skin graft take have been performed by many investigators and it has been shown that skin grafts become viable after revascularization from the graft bed after passing through a certain period of circulatory interruption. However, there are some disagreements between researchers regarding the role played by serum and the onset time of revascularization. We have investigated the changes in adenosine 5'-triphosphate (ATP) and glucose levels of skin grafts in rats to understand the biochemical process of skin graft take. A total of 250 male Wistar rats were used, and 450-microns split-thickness skin grafts were cut from their backs using a dermatome. In Group 1, the skin graft was grafted onto the dorsal fascia of the same rat. In Group 2, a 191-microns-thick Millipore filter with 1.5-microns pores was interposed between the graft and the dorsal fascia to inhibit revascularization. In Group 3, the skin graft was enveloped in a piece of gauze containing physiological saline solution and incubated at 37 degrees C. Skin grafts were removed at 6, 12, and 24 hours as well as on days 2, 3, 4, 5, 6, and 7 after grafting. The ATP and glucose levels were extracted from the grafts and quantitated using high-performance liquid chromatography. In Group 1, the ATP and glucose levels in the graft decreased rapidly after grafting; the ATP level fell to approximately 30% of that before grafting and glucose to about 20%, on days 2 and 3, respectively. Thereafter, these levels increased gradually.(ABSTRACT TRUNCATED AT 250 WORDS)


Estradiol regulates class I alcohol dehydrogenase gene expression in renal medulla of male rats by a post-transcriptional mechanism. Rat kidney contains alcohol dehydrogenase (ADH) activity which appears to be identical to the class I ADH expressed in liver. Treatment of male rats with estradiol for 10 days induced ADH activity and protein in the kidney approximately 3-fold. This was not the result of suppression of testosterone levels by estrogen, as castration did not increase ADH activity. In situ hybridization of kidney sections showed that ADH transcripts were localized to the medulla, that the basal level of mRNA is very low in the male, and that the induction of ADH mRNA by estradiol was approximately 10-fold. As estimated from Northern blot analysis, the induction of the mRNA was approximately 7-fold. Thus, induction of ADH mRNA substantially exceeded the increase of ADH activity and protein. Since the estradiol-treated rats lost weight relative to the oil-injected controls, the effect of starvation on ADH mRNA in kidney was examined. Starvation decreased kidney ADH activity by about 30% but increased mRNA about 2-fold. Time course experiments demonstrated induction of ADH mRNA by estradiol within 1 h with the maximum level achieved by 24 h. The transcription rate of the ADH gene as assessed by nuclear run-on assays performed at 1 and 24 h after treatment with estradiol was unchanged. We conclude that estradiol induces ADH mRNA in kidney by a post-transcriptional mechanism.


Two coding regions closely linked to the rat apolipoprotein E gene: nucleotide sequences of rat apolipoprotein C-I and ECL cDNA. Restriction fragments isolated from a 17-kb rat genomic DNA clone containing the gene for apolipoprotein (apo) E were radiolabeled and used to screen a rat liver cDNA library. A cDNA clone hybridizing to a 6-kb genomic DNA fragment was isolated and the nucleotide sequence of the cDNA insert determined. The sequence was homologous to the sequence for human apo C-I and was used to derive the corresponding amino acid sequence. Unlike human apo C-I, mature rat apo C-I contains histidine, lacks valine, and has alanine at the C terminus and aspartate as the N terminus. Screening the rat liver cDNA library with a radiolabeled 1.9-kb restriction fragment from the genomic DNA clone containing the rat apo E gene identified another cDNA clone (ECL cDNA). Nucleotide sequencing yielded a derived 75-amino-acid sequence for the ECL protein with a hydrophobicity profile similar to that of rat apo C-I. Northern analysis demonstrated a 0.50-kb band for ECL mRNA. The tissue-specific expression of the gene is similar to that of rat apo C-I. This study indicates that the rat apo C-I and ECL genes are closely linked, about 4.5 and 12 kb downstream of the apo E gene, respectively.


Phosphatidic acid and lysophosphatidic acid stimulate receptor-regulated membrane currents in the Xenopus laevis oocyte. External application of dioleoyl-phosphatidic acid and oleoyl-lysophosphatidic acid stimulated Ca(2+)-dependent chloride currents in voltage-clamped Xenopus laevis oocytes. The responses were observed in oocytes from which follicular cells had been removed, indicating they were intrinsic to the oocyte itself. The lipid-induced Ca(2+)-dependent chloride currents were observed in the absence of extracellular calcium, were blocked by intracellular injection of the calcium chelator, bis(O-aminophenoxy)-ethane N,N,N'N'-tetraacetic acid, and could not be elicited by direct intracellular injection of the active lipids. The thresholds for dose-dependent current responses to dioleoyl-phosphatidic acid (100 nM) and for oleoyl-lysophosphatidic acid (10 nM) indicated that the lipid activities on oocytes were potent. With repeated or prolonged administration of either active lipid, responses exhibited desensitization. These results demonstrate that the Xenopus oocyte expresses endogenous functional responses for the mitogenic lipids phosphatidic acid and lysophosphatidic acid and thus provides a powerful model for characterization of the pharmacology and transduction pathways of these responses.


A bullous skin disease patient with autoantibodies against separate epitopes in 1 mol/L sodium chloride split skin. We describe a patient with a subepidermal bullous skin disease associated with autoantibodies recognizing separate epitopes in 1 mol/L sodium chloride (NaCl) split skin. Direct immunofluorescence microscopy showed deposits of immunoglobulins and C3 in a continuous pattern in the patient's epidermal basement membrane zone. Direct immunoelectron microscopy demonstrated thick deposits of IgG overlying the lamina lucida and the lamina densa in a unique pattern. The patient had circulating IgG anti-basement membrane zone antibodies that bound both sides of 1 mol/L NaCl split skin, exhibited at least a fourfold-higher titer against the dermal side of this test substrate, and bound basal keratinocyte hemidesmosomes as well as focal sites along the superior portion of the lamina densa on indirect immunoelectron microscopy. Affinity purification of anti-basement membrane zone antibodies against epidermal or dermal strips of 1 mol/L NaCl split skin yielded IgG that only bound the side of split skin from which it was eluted. The patient's serum contained IgG that immunoprecipitated and immunoblotted the 230- and 170-kd bullous pemphigoid antigens. Affinity purification of patient antibody against bullous pemphigoid antigen immobilized on nitrocellulose paper yielded IgG that bound only the epidermal side of 1 mol/L NaCl split skin. The patient showed no evidence of reactivity against type VII collagen by direct immunoelectron microscopy, indirect immunoelectron microscopy, or immunoblot. This patient's bullous skin disease is associated with IgG anti-basement membrane zone antibodies with two specificities: one recognizing the bullous pemphigoid antigen in the epidermal side of 1 mol/L NaCl split skin, and another binding a distinct, yet presently unidentified, epitope in the superior aspect of the lamina densa.


Metastatic nasopharyngeal carcinoma initially presenting as cervical lymphadenopathy. A report of two cases that resembled Hodgkin's disease. We describe two patients with nasopharyngeal carcinoma who initially presented with cervical lymphadenopathy. Lymph node biopsy specimens in each patient were initially diagnosed as Hodgkin's disease. In both cases, the neoplastic cells had large, vesicular nuclei with prominent eosinophilic nucleoli; some neoplastic cells were identified in lacunar spaces. In addition, numerous inflammatory cells were present, including eosinophils, lymphocytes, and plasma cells. At the time of referral, the correct diagnosis of metastatic carcinoma was made, and primary nasopharyngeal carcinomas were subsequently identified. The possibility of metastatic nasopharyngeal carcinoma should always be considered in adults with enlarged cervical lymph nodes that resemble Hodgkin's disease. The cytologic features of the malignant cells are the clue to the correct diagnosis. Immunophenotypic studies easily resolve this diagnostic dilemma if the possibility of metastatic nasopharyngeal carcinoma is considered.


Cardiovascular cyclic nucleotide phosphodiesterases and their role in regulating cardiovascular function. We have described five phosphodiesterase (PDE) isozymes that can be found in cardiac and vascular smooth muscle of animals and humans. Much of the evidence for the role that these isozymes have in the regulation of cellular processes has been generated through, or awaits, the identification of selective and potent PDE inhibitors. While selective inhibitors of the cGMP-inhibitable (cGi)-PDE isozyme have been approved for use in the acute treatment of heart failure, selective inhibitors of the cGMP-PDE have not been extensively explored as potential candidates for the treatment of cardiovascular diseases. More potent selective inhibitors of the cGMP-PDE isozyme are needed to determine whether these pharmacological potentiators of EDRF and ANP will be useful in the therapy of angina, hypertension or heart failure.


[The effect of a restriction of the feed intake on the composition of the blood and on the development and composition of different tissues of sheep during growth. 2. The development of different tissues and the concentration of protein, DNA and RNA]. The concentration of protein in the cerebrum of sheep after a period of feed restriction (group 2) was lower (94.5 +/- 10.2) than in normally fed sheep of group 4 (101.4 +/- 9.4 mg/g wet weight). In the group 2 the concentration of protein in the M. longissimus dorsi and in the M. semimembranosus was also smaller. A high DNA-concentration was determined in the intestinal lymph nodes, in the spleen and in the lung. The DNA-concentration of the testes of group 2 (7.17 +/- 2.92) was higher than that of the group 4 (4.46 +/- 1.70 mg/g w. w.), also that of the renal fat tissue (0.39 +/- 0.18 resp. 0.20 +/- 0.09). The highest protein: DNA-relation in group 4 was found in the fat tissue (203.5: 1) and the lowest in the spleen (15.3: 1). A high RNA-concentration was analysed in the lymph nodes, in the spleen and in the lung. The RNA-concentration in the fat tissue of group 2 (0.34 +/- 0.13) was higher that that in group 4 (0.15 +/- 0.08 mg/g w. w.).


Interferons induce xanthine dehydrogenase gene expression in L929 cells. Human interferon-alpha A/D (Bg/II) (IFN-alpha A/D) and mouse interferon-gamma (IFN-gamma) are shown to induce xanthine dehydrogenase (XD) mRNA in L929 fibroblastic cells. XD mRNA accumulation after IFN-alpha A/D treatment is relatively fast, being already evident after 4 h and reaching its maximum after 24 h. IFN-alpha A/D is active in inducing XD mRNA at 0.1 unit/ml and it is maximally active at 10(3) units/ml. The half-life of the XD message is unaffected by IFN-alpha A/D treatment, whereas the transcriptional activity of the XD gene and the concentrations of XD heterogeneous nuclear RNA are increased by 2- and 6-fold respectively. The effect of IFN-alpha A/D on XD mRNA is insensitive to cycloheximide, suggesting that protein synthesis de novo is not required. Experiments conducted with specific inhibitors suggest that protein kinase C, cyclic AMP and arachidonic acid metabolites derived from lipoxygenase or cyclooxygenase do not act as second-messenger molecules in the induction of XD mRNA by IFN-alpha A/D. XD mRNA is also induced in NIH3T3 fibroblastic cells, but not in F9 teratocarcinoma or B16 melanoma cells after treatment with IFN-alpha A/D. NIH3T3 are the only cells so far tested that have detectable XD and xanthine oxidase activities under basal conditions and after IFN-alpha A/D treatment, although their responsiveness to the cytokine is much less than that observed in L929 cells.


Renaturation and partial peptide sequencing of mitogen-activated protein kinase (MAP kinase) activator from rabbit skeletal muscle. Mitogen-activated protein kinase (MAP kinase) activator was purified 2000-fold from skeletal muscle, and proteins which co-purified with the activator were analysed after SDS/PAGE by renaturation and partial sequencing. Activity for tyrosine and threonine phosphorylation of MAP kinase was present in two bands of approx. 48 and 46 kDa, which have sequence similarity to small GTP-binding protein p25 GDP dissociation inhibitor and protein kinases (PBS2, SPK1+, STE7, BYR1) respectively.


Changes in keratin expression during fetal and postnatal development of intestinal epithelial cells. We have investigated keratin expression in fetal, newborn and adult rat intestines by immunofluorescence staining, immunoblotting of two-dimensional gels and Northern blot analysis of total cellular RNAs. Keratin-type intermediate filaments, composed predominantly of keratin no. 19, were observed already in the undifferentiated stratified epithelium present at 15-16 days of gestation. The marked maturation and differentiation of the epithelium taking place at 18-19 days of gestation was characterized by the appearance of the differentiation-specific keratin no. 21 and by a significant increase in the relative amount of keratin no. 8. The keratin pattern typical of adult villus cells became established at the time of birth, and was marked by a considerable increase in the complexity of the keratin-related polypeptides detected on two-dimensional gels, indicative of extensive post-translational modification of all keratins. Starting at 20 days of gestation there was a major increase in the relative abundance of mRNAs coding for keratin nos. 8, 19 and 21; in contrast, the relative amount of keratin no. 18 mRNA reached a peak shortly after birth and declined to very low levels in adult intestine. These results demonstrated marked changes in keratin expression and post-translational processing taking place at key stages of intestinal development. The appearance of keratin no. 21 in coincidence with the formation of an adult-type brush border and terminal web would be consistent with it having an important role in the organization of the intermediate filament network in the apical cytoplasm of the differentiated intestinal cells.


The rat angiotensin II AT1A receptor couples with three different signal transduction pathways. To examine whether the subpopulation of the rat type 1 angiotensin II (AII) receptor (AT1A) couples with a single or multiple signal transduction pathways, we constructed Chinese hamster ovary (CHO) cell lines producing the recombinant receptor. The expressed AT1A receptor exhibits typical pharmacological characteristics of the AT1 receptor, known to mediate the main physiological function of AII. Addition of AII to the CHO cells induced a rapid, transient increase in intracellular free Ca2+ concentrations ([Ca2+]i) followed by a lower, sustained phase. Nicardipine, a blocker of voltage-dependent L-type Ca2+ channels, attenuated the transient [Ca2+]i response and abolished the sustained phase. The transient phase was also reduced dose-dependently by the phospholipase C inhibitor neomycin. Furthermore, AII inhibited forskolin-evoked cAMP accumulation. These data suggest, although another subpopulation named AT1B is present, that the rat AT1A receptor can independently couple with all three signal transduction pathways known to be induced by AII: i.e., i) activation of phospholipase C resulting in InsP3 generation with a subsequent release of intracellularly stored Ca2+, ii) activation of dihydropyridine-sensitive voltage-dependent Ca2+ channels, and iii) inhibition of adenylate cyclase activity.


A plasminogen-related gene is expressed in cancer cells. The breakdown of blood clots requires the fibrinolytic action of the serine proteinase plasmin, a two-chain polypeptide derived posttranslationally from its precursor zymogen, plasminogen. While investigating plasminogen gene expression in human extrahepatic tissues, a cDNA sequence was obtained which closely resembled the plasminogen cDNA, yet appeared to represent a distinct gene product. This sequence, which represents the transcript of the recently characterized plasminogen-related gene B, encodes a putative polypeptide of Mr 8800 and is expressed most prominently in malignant cancer cells.


Hammerhead ribozyme cleavage of hamster prion pre-mRNA in complex cell-free model systems. The cleavage properties of a trans-acting hammerhead ribozyme targeted 51 bases upstream of the putative splicing branch point in the hamster prion pre-mRNA intron were investigated in cell-free model systems in vitro. The specificity of cleavage was demonstrated by the inability of this ribozyme to cleave a non-homologous synthetic message encoding part of the beta amyloid peptide precursor, beta APP, and by the inability of the prion pre-mRNA to be cleaved by a ribozyme targeted to beta amyloid peptide precursor mRNA. Also, the addition of total RNA isolated from rat brain had only a minimal effect on the cleavage of the prion substrate pre-mRNA by the ribozyme. Finally neither the presence of 100 ng of nuclear or cytoplasmic proteins were found to affect the rate of cleavage in vitro.


Leukocyte chemotactic activity of FKBP and inhibition by FK506. Cyclophilin, the cyclosporin A binding protein and member of the immunophilin family of proteins, demonstrates leukocyte chemotactic activity. In this study we demonstrate that FKBP, the FK506 and rapamycin binding protein, also displays leukocyte chemotactic activity. The chemotactic activity of FKBP is inhibited by FK506, however, FK506 was unable to inhibit cyclophilin-stimulated chemotactic activity. Rapamycin was unable to prevent the chemotactic activity of FKBP, similarly, the CsA analogue Me6Ala-CsA while displaying cyclophilin binding was unable to block cyclophilin-stimulated chemotactic activity. These results suggest that in addition to their intracellular role the immunophilins may also function as chemotactic agents, furthermore this activity is modulated by immunosuppressants.


Porin of Pseudomonas aeruginosa forms low conductance ion channel in planar lipid bilayers. Protein E1, a porin of the outer membrane of Pseudomonas aeruginosa, was reconstituted into planar lipid bilayers. Single channel conductance of the protein appeared to be 230 pS (pico siemens) in 1 M KCl-10 mM Hepes, pH7.2. This value is approximately 5 times lower than the conductance of the OmpF channel of Escherichia coli. Conductance increased linearly as the membrane potential was raised from -200 mV to +200 mV, and was nearly proportional to the KCl concentration. These results show that protein E1 is probably a genuine porin in the P. aeruginosa outer membrane supporting the earlier conclusion that protein E1 forms a small channel.


Interleukin-8 inhibits the induction of nitric oxide synthase in rat peritoneal neutrophils. The effect of interleukin(IL)-8 and leukotriene B4 (LTB4) on the induction of nitric oxide (NO) synthase in rat peritoneal neutrophils (PMN) ex vivo was studied. IL-8, but not LTB4, caused concentration-dependent inhibition of the induction of a Ca(2+)-independent NO synthase ex vivo. The effect of IL-8 was not attributable to the synthesis of an inhibitor of this enzyme. These findings suggest complex regulatory control of the induction of NO synthase by cytokines.


Species heterogeneity of hepatic alpha 1-adrenoceptors: alpha 1A-, alpha 1B- and alpha 1C-subtypes. alpha 1-Adrenergic activation stimulated phosphorylase and phosphoinositide turnover in hepatocytes from guinea pigs, rats and rabbits. Chlorethylclonidine inhibited these effects in rat and rabbit cells but not in guinea pig hepatocytes; low concentrations of 5-methyl urapidil blocked the alpha 1 actions in guinea pig and rabbit liver cells, but not in rat hepatocytes. Binding competition experiments also showed high affinity for 5-methyl urapidil in liver membranes from guinea pigs and rabbits and low affinity in those from rats. The data indicated that guinea pig hepatocytes express alpha 1A-, rat hepatocytes alpha 1B- and rabbit hepatocytes alpha 1C- adrenoceptors. This was confirmed by Northern analysis using receptor subtype-selective probes.