The movement of fluorescent endocytic tracers in Plasmodium falciparum infected erythrocytes.

The fluorescent lipophilic probe 1,1'-dihexadecyl-3-3'-3-3'- tetramethylindocarbocyanine (diIC16) inserted in the red cell surface, functioned as a non-exchangeable lipid marker which was not metabolised or toxic in plasmodial cultures. Invasion by Plasmodium falciparum resulted in the internalisation of the lipid, suggesting the uptake of red cell membrane components during parasite entry. The fluorescent lipid was not transported from red cell to parasite membranes at subsequent stages of development, but label in the erythrocyte-derived parasitophorous vacuole was eventually detected in daughter merozoites. Fluorescent dextrans of 10 kDa in the extracellular medium were also not internalised during intraerythrocytic parasite growth. The results support that with the exception of the invasion step, plasmodial infection does not induce endocytosis in the erythrocyte membrane. Despite the lack of endocytosis, both D and L stereoisomers of the head group blocked phospholipid analogue N-4-nitrobenzo-2-oxa-1,3-diazoledipalmitoyl phosphatidylethanolamine (N-NBD-PE) inserted in the erythrocyte membrane, were internalised by mature infected erythrocytes. Lipid internalisation occurred by a non head group dependent parasite mechanism, which could account for the stage-specific uptake of phospholipids observed in mature infected erythrocytes. We were unable to detect the transport of carbocyanine dyes and N-NBD-PE from intracellular parasites back to the erythrocyte membrane. Additionally, the carbocyanine dyes were not transferred between adjacent organisms in a double infected red cell. The data argue for the absence of bulk membrane lipid transport between individual parasites and their host cell bilayer in an infected erythrocyte.


[Spinal epidural stimulation for central pain caused by spinal cord lesion].

Epidural spinal cord stimulation was carried out in 4 patients with denervation caused by spinal cord lesion, and we reviewed previously reported cases. Initial result showed at 1 week in 100% of our cases, but about 1/3 of the cases, even those with the same denervation caused by spinal cord lesion, had no pain relief at this stage in previously reported cases. In our cases, excellent pain relief was gained temporarily, even though the painful area and the spinal cord lesion were separated somatotopically in 2 cases (case 3, 4). Temporary success bore no relationship to quality and duration of pain. In all cases except case 1, a rapidly decreasing effectiveness was noted, and finally no pain relief was gained at all after 4, 3 and 5 months, respectively. In case 1 there was persistent pain relief estimated at 70-80% after 19 months, only when the spinal cord was stimulated. Epidural stimulation also produced sensations in the painful area. Spinal cord stimulation would suppress at least the dorsal horn neurons which were destroyed by various kinds of diseases. A decline in effectiveness with time would occur due to essential causes of the deafferentation pain, such as anatomical and regeneration factors.


[AFP producing gastric cancer manifested by metastasis to the tentorium cerebelli; case report and review of the literature].

We report a case of AFP producing gastric cancer manifested by metastasis to the tentorium cerebelli. A 66-year-old male patient was admitted with dysarthria, occipital headache and nausea on May 1, 1990. Neurological examination revealed signs of increased intracranial pressure and the right-sided cerebellar hemispheric signs. CT and MRI showed a round tumor shadow 3cm in diameter, which originated in the right-side tentorium cerebelli and grew in the posterior fossa. Tumor stains fed by the right tentorial artery were recognized by angiography. Serum AFP level was 503.5ng/ml. The patient underwent an operation under general anesthesia in the prone position. The tumor was totally removed via the suboccipital transtentorial approach. Histological examination revealed AFP producing adenocarcinoma. The patient was found to have a gastric cancer after neurosurgical operation, and underwent subtotal gastrectomy by surgeons. Serum AFP level was 254.5ng/ml after removal of metastatic brain tumor, and 5.0ng/ml after subtotal gastrectomy.


Anticonvulsant role of nigrotectal projection in the maximal electroshock model of epilepsy--II. Pathways from substantia nigra pars lateralis and adjacent peripeduncular area to the dorsal midbrain.

Lesion evidence suggests that the superior colliculus is essential for mediating the anticonvulsant properties of nigral suppression in the electroshock model of epilepsy. However, our companion paper [Redgrave et al. (1991) Neuroscience 46, 379-390] established that the region of dorsal midbrain where bicuculline was most effective in suppressing tonic hindlimb extension did not correspond well with the known distribution of nigrotectal terminals. The purpose of the present anatomical study was, therefore, to investigate in more detail ventral midbrain connections to the dorsal midbrain anticonvulsant zone in rat. Small injections (10-20 nl) of a 1% solution of wheatgerm agglutinin conjugated with horseradish peroxidase were made specifically into the region of dorsal midbrain where bicuculline was maximally effective. Numerous retrogradely labelled cells were found in substantia nigra pars lateralis and adjacent peripeduncular area but not in substantia nigra pars reticulata. Retrogradely labelled cells were also located in ventral zona incerta. When wheatgerm agglutinin-horseradish peroxidase injections were made into lateral substantia nigra, a region of anterogradely transported reaction product characteristic of nerve terminals was observed in the caudolateral deep layers and underlying reticular tissue; this area corresponded well to the dorsal midbrain anticonvulsant zone. These data suggest that, in the electroshock model of epilepsy, direct connections between substantia nigra pars lateralis and adjacent peripeduncular area and the dorsal midbrain anticonvulsant zone could be critical for mediating the anticonvulsant properties previously attributed to substantia nigra pars reticulata. During the course of this study, anterograde projections from substantia nigra pars lateralis and adjacent peripeduncular area to both superficial and intermediate layers of the ipsilateral superior colliculus were noted. Additional experiments using retrograde transport of the fluorescent tracer Fast Blue confirmed these projections.


Increased S100 beta neurotrophic activity in Alzheimer's disease temporal lobe.

The confirming diagnosis of Alzheimer's disease includes an assessment of the concentration of neuritic plaques in the temporal lobe of the brain. The presence of abnormal levels of neurotrophic factors in Alzheimer's disease is one possible explanation for the increased concentration of aggregates of overgrown neurites in the neuritic plaques of Alzheimer's disease. The protein S100 beta, a neurotrophic factor produced by astroglia in the brain, induces neurite outgrowth in cerebral cortical neurons. The generation of specific S100 beta antibodies, the cloning of a full-length cDNA encoding the S100 beta mRNA, and the development of a neurite extension assay system for S100 beta allowed testing of the hypothesis that Alzheimer's disease S100 beta expression is elevated in brain temporal lobe where neuritic plaques are concentrated. The levels of S100 beta protein, mRNA, and specific neurotrophic activity were elevated 10-20-fold in extracts of temporal lobe from autopsy samples of Alzheimer's disease patients compared to those of aged control patients. The cells containing the increased S100 beta were reactive astrocytes; the neuritic plaques were surrounded by S100 beta-containing astrocytes. The elevated levels of S100 beta provides a link between the prominent reactive gliosis and neuritic plaque formation in this common disease of the elderly and raises the possibility that S100 beta contributes to Alzheimer's disease neuropathology.


Significance of decreased lumbar CSF levels of HVA and 5-HIAA in Alzheimer's disease.

The monoamine metabolites homovanillic acid (HVA), 5-hydroxy-indoleacetic acid (5-HIAA) and 4-hydroxy-3-methoxy-phenylglycol (HMPG) were determined in lumbar cerebrospinal fluid (CSF) of 123 patients with Alzheimer's disease (AD) and 57 healthy controls. Despite CSF sampling under strictly standardized conditions, a wide variability in values among both patients and controls was found, as well as fluctuations in repeated samples from individual patients. This suggests that several unknown factors influence the lumbar CSF levels of monoamine metabolites. The AD group showed significantly lower mean levels of HVA (p less than 0.0001) and 5-HIAA (p less than 0.0001) than the control group. A relation between severity of disease and HVA was also found. The widespread neurotransmitter disturbance in AD, together with the nonspecificity of reduced lumbar HVA and 5-HIAA levels, suggests that the changes are nonspecific, secondary to the cerebral degeneration in AD.


Serotonin synthesis in adrenochromaffin cells.

The inter-renal (adrenal) gland of amphibians is composed of chromaffin and steroidogenic cells which can interact through a paracrine mode of communication. We have previously shown that serotonin is present in secretory granules of frog adrenochromaffin cells; concurrently, we have demonstrated that serotonin is a potent stimulator of corticosterone and aldosterone secretion by adrenocortical cells. The aim of the present study was to determine the origin of the amine contained in frog chromaffin cells. Using 3H-labelled tryptophan as a precursor, we observed the formation of substantial amounts of serotonin and its metabolite 5-hydroxyindoleacetic acid by frog inter-renal slices. Newly synthesized serotonin was secreted into the incubation medium and the release process was enhanced by depolarizing concentrations of KCl. Fluoxetine, and inhibitor of serotonin uptake, caused an increase of 3H-labelled serotonin in the incubation medium, suggesting that the indoleamine was taken up again by adrenal chromaffin cells. The capacity of the frog inter-renal gland to synthesize serotonin was also demonstrated by incubating inter-renal slices with non-labelled tryptophan or 5-hydroxytryptophan. In these conditions, we observed that the rate of synthesis was higher when 5-hydroxytryptophan was used as a a precursor, rather than tryptophan. Taken together, these results indicate that chromaffin cells, which have the capacity for synthesizing and releasing serotonin, behave like authentic serotonergic paraneurons. As far as is known, these data provide the first evidence for the occurrence of tryptophan-5-hydroxylase activity within the adrenal gland.


Histochemical mapping of nitric oxide synthase in the rat brain.

The NADPH-diaphorase histochemical technique provides a simple and robust method to stain select populations of neurons throughout the brain. We have recently identified the enzyme responsible for this histochemical reaction to be nitric oxide synthase. This enzyme is responsible for the calcium-dependent synthesis of nitric oxide from arginine. Nitric oxide acts as a novel neural messenger by stimulating soluble guanylyl cyclase thereby increasing the levels of cyclic guanosine 3',5'-monophosphate in target cells. Thus the NADPH-diaphorase histochemical method allows the direct visualization of the neurons which use this novel signal transduction pathway. We now describe the detailed distribution of this enzyme in the rat brain. Our results suggest a widespread role for the nitric oxide-cyclic guanosine monophosphate system in the nervous system.


Potential- and acetylcholine-activated ionic currents of pheochromocytoma PC12 cells during incubation with nerve growth factor.

Pheochromocytoma PC12 cells incubated with and without nerve growth factor were investigated using the patch-clamp technique in the whole-cell recording mode, and the concentration clamp method in the rat. On the fourth day of incubation with nerve growth factor, sodium potential-activated ionic currents appeared in the membranes of the most morphologically differentiated cells. At the same period a three-fold increase of acetylcholine-activated current density, compared with the cells incubated without nerve growth factor, was observed. Thus, the qualitative and quantitative changes in membrane properties can be a result of metabolic reorganization in PC12 cells induced by nerve growth factor and accompanied by morphological differentiation according to neuronal phenotype.


Study of the topographical distribution of different populations of motoneurons within rat's nucleus ambiguus, by means of four different fluorochromes.

The topographical neuronal distribution within the rat nucleus ambiguus has been studied with the simultaneous retrograde labeling technique by means of four different fluorochromes injected within the various muscles and/or nerves of the oro-pharyngeal region. This technique has permitted the identification of several types of neurons along the same coronal plane. Most were motoneurons innervating the various muscles of the upper airway, including pharyngeal constrictor, stylopharyngeal, intrinsic laryngeal and the upper portion of the esophagus. Some neurons may have been preganglionic parasympathetic neurons. No evidence of axonal branching of any of the labeled motoneurons or parasympathetic neurons was found.


Injury-induced reduction of acidic fibroblast growth factor levels in the distal parts of rat sciatic nerve.

Acidic fibroblast growth factor (aFGF) level in sciatic nerve after lesioning was measured by enzyme immunoassay to determine if aFGF functions as a neurotrophic factor like nerve growth factor (NGF). Whereas the NGF level increased in distal segments, the aFGF level there decreased after transection or crushing and recovered to the original level by 10 weeks after crushing. The amount of aFGF mRNA in the sciatic nerve was extremely low to supply the high level of protein found in the sciatic nerve. Sympathetic ganglia, dorsal root ganglia, and spinal cord, which contain neuronal cell bodies extending their axons into the sciatic nerve, showed a greater or similar level of aFGF as sciatic nerve. These results imply that aFGF is synthesized in neuronal cell bodies and distributed anterogradely into their axons. Difference of injury-induced changes in levels between aFGF and NGF suggests distinct mechanisms of the effects elicited from these factors on regeneration of the sciatic nerve.


In vitro interaction between cerebellar astrocytes and granule cells: a putative role for nitric oxide.

In primary cultures of astrocytes and granule cells from neonatal rat cerebellum, the activity and function of nitric oxide (NO) synthase were measured by the conversion of [3H]arginine to [3H]citrulline and the accumulation of cyclic guanosine monophosphate (cGMP), respectively. The glutamate receptor agonist N-methyl-D-aspartate (NMDA) and the Ca2+ ionophore A23187 stimulated NO synthase activity in cerebellar granule cells but not in astrocytes. In granule cells, NMDA, A23187, and sodium nitroprusside (SNP) elicited an accumulation of cGMP, whereas only SNP was active in astrocytes. However, in astrocytes that were incubated together with granule cells, NMDA induced a more than 3-fold increase in the concentration of cGMP; this increase was blocked by both the NO synthase inhibitor NG-monomethyl-L-arginine (MeArg) and the allosteric NMDA receptor antagonist (+)5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate (MK-801). Thus, cerebellar astrocytes do not appear to express NO synthase but do contain guanylate cyclase, which can be activated by an NO-like factor produced by cerebellar granule cells after stimulation by NMDA.


The organisation of fibres within the rat basis pedunculi.

The organisation of corticofugal fibres within the basis pedunculi of rats was studied using wheat germ agglutinin-horseradish peroxidase as an orthograde tracer. Following cortical injections, labelled fibres were distributed within the cerebral peduncle in an orderly way. Fibres which originate from cells in the frontal cortex maintain a position in the ventromedial part of the basis pedunculi. Fibres from the occipital and temporal cortex travel in the most dorsolateral part. Somatosensory fibres travel between these two. The extent of labelled fibres within the peduncles is correlated with the relative density of corticopontine cells arising from different areas of the cerebral cortex.


Appearance of paired nucleated, Tau-positive glia in patients with progressive supranuclear palsy brain tissue.

Many Tau-positive glia with paired nuclei and astrocyte type morphology were identified in three brains from patients with progressive supranuclear palsy (PSP). They were positive by Bielschowsky's and Bodian's silver staining as well as by immunostaining with Tau-2, Alz-50, anti-GFAP and anti-paired helical filament antibodies, but not with anti-ubiquitin antibody. They were predominantly localized in the striatum, thalamus and frontal cortex but were not seen in white matter and were not plentiful in areas of heavy neuronal degeneration. Electron microscopy clearly showed the nuclear pairing and localized the Tau protein to bundles suggestive of microtubules in the cytoplasm and proximal processes. Such glial cells were rarely seen in cases of other neurodegenerative diseases or neurologically normal controls. These data suggest that there is an unusual gliotic reaction in PSP in brain areas which show relatively little neuronal loss.


[Quantitative morpho- and densitometric parameters in the evaluation of aspiration cytology smears of the breast].

Quantitative morpho- and densitometric analysis of Feulgen stained breast aspiration smears were performed by absorption cytophotometer and a TV image analyser. The DNA Index, and the ratio of G1-S-G2 phase fraction ratio yielded by the quantitative DNA analysis, showed significant differences between benign and malignant alterations. Based on the 13 morphometric and 5 densitometric parameters the TV-image analyser could classify the examined smears 80% correctly by cluster analysis. Groups of the cytological Grade I.-III. were also differentiable by this method. The application of TV image analyser system in the diagnosis of aspiration cytological smears could be suggested. The complex quantitative morpho- and densitometric analysis could contribute to the correct classification of diploid tumours.


Genomic organisation and expression of a differentially-regulated gene family from Leishmania major.

We have isolated and characterised a differentially-regulated gene family in the protozoan parasite Leishmania major. The family contains 5 genes linked within a 10Kb region of the genome: three of the genes are closely related in DNA sequence, the other two have only limited homology. Post-transcriptional control of the differential expression pattern is suggested by detection of precursor RNA molecules containing intergenic sequences and evidence that mature mRNA molecules contain a 35nt spliced leader sequence at their 5' ends. These features support a model of polycistronic transcription in which the stability and differential processing of precursor RNA molecules determine the steady state levels of mature mRNA. We have identified several DNA sequence motifs within the gene family that have potential roles in differential processing and/or RNA stability: an alternative 5' splice acceptor site for trans-splicing; a putative polyadenylation site; and a region of potential secondary structure within 3' flanking sequences. The 3' sequence elements are conserved in those genes that share the same pattern of differential regulation. To our knowledge, this is the first example of coordinated differential-regulation of a non-identical gene cluster in Leishmania.


Increased specificity for antisense oligodeoxynucleotide targeting of RNA cleavage by RNase H using chimeric methylphosphonodiester/phosphodiester structures.

One of the inherent problems in the use of antisense oligodeoxynucleotides to ablate gene expression in cell cultures is that the stringency of hybridization in vivo is not subject to control and may be sub-optimal. Consequently, phosphodiester or phosphorothioate antisense effectors and non-targeted cellular RNA may form partial hybrids which are substrates for RNase H. Such processes could promote the sequence dependent inappropriate effects recently reported in the literature. We have attempted to resolve this problem by using chimeric methylphosphonodiester/phosphodiester oligodeoxynucleotides. In contrast to the extensive RNA degradation observed with all-phosphodiester oligodeoxynucleotides, highly modified chimeric antisense effectors displayed negligible, or undetectable, cleavage at non-target sites without significantly impaired activity at the target site. We also note that all of the all-phosphodiester oligodeoxynucleotides tested demonstrated inappropriate effects, and that such undesirable activity could vary widely between different sequences.


Formation of phosphonester bonds catalyzed by DNA polymerase.

3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma- diphosphate and 2'-deoxythymidine-5'-(alpha-methylphosphonyl)-beta, gamma- diphosphate have been synthesized. Both compounds are incorporated into DNA chains during catalysis by reverse transcriptases of human immunodeficiency (HIV) and avian myeloblastosis (AMV) viruses, DNA polymerase beta from rat liver, terminal deoxynucleotidyl transferase from calf thymus and (at a very low rate) is by E. coli DNA polymerase I, Klenow fragment. The first compound is a termination substrate while the second is capable of multiple incorporation into the DNA chains. For instance, reverse transcriptase catalysis resulted in the appearance of 8 residues of second compound. DNA polymerases alpha and epsilon from human placenta incorporated none of the above compounds into DNA chains, although an inhibition of DNA synthesis by both compounds was observed with all enzymes mentioned. The 3'----5'-exonuclease activity of DNA polymerase I, Klenow fragment, hydrolyzed DNA fragments containing phosphonomethyl internucleoside groups, while such DNA fragments were resistant to the E. coli exonuclease III.


Thermodynamic parameters for loop formation in RNA and DNA hairpin tetraloops.

We determined the melting temperatures (Tm) and thermodynamic parameters of 15 RNA and 19 DNA hairpins at 1 M NaCl, 0.01 M sodium phosphate, 0.1 mM EDTA, at pH 7. All these hairpins have loops of four bases, the most common loop size in 16S and 23S ribosomal RNAs. The RNA hairpins varied in loop sequence, loop-closing base pair (A.U, C.G, or G.C), base sequence of the stem, and stem size (four or five base pairs). The DNA hairpins varied in loop sequence, loop-closing base pair (C.G, or G.C), and base sequence of the four base-pair stem. Thermodynamic properties of a hairpin may be represented by nearest-neighbor interactions of the stem plus contributions from the loop. Thus, we obtained thermodynamic parameters for the formation of RNA and DNA tetraloops. For the tetraloops we studied, a free energy of loop formation (at 37 degrees C) of about +3 kcal/mol is most common for either RNA or DNA. There are extra stable loops with delta G degrees 37 near +1 kcal/mol, but the sequences are not necessarily the same for RNA and DNA. The closing base pair is also important; changing from C.G to G.C lowered the stability of several tetraloops in both RNA and DNA. These values will be useful in predicting RNA and DNA secondary structures.


Processing in the 5' region of the pnp transcript facilitates the site-specific endonucleolytic cleavages of mRNA.

The primary transcript of pnp, the gene encoding polynucleotide phosphorylase in Escherichia coli, is processed in the 5' end region by ribonuclease III (RNase III). The unprocessed transcript shows enhanced stability compared with the processed transcript. We report here that, unlike the processed transcript, the unprocessed pnp transcript did not accept endonucleolytic attack at, at least, five cleavage sites. Sequencing analysis of the four cleavage products shows no sequence specific to all these sites, but AU rich stretches were observed at three sites.


The primary transcription unit of the human alpha 2 globin gene defined by quantitative RT/PCR.

We have set up an experimental system to map the primary transcription unit of the human alpha 2 globin gene. The duplicated human alpha globin genes (alpha 2-alpha 1) were linked to the alpha globin locus Positive Regulatory Element (PRE) and stably transfected into murine erythroleukaemia cells. We then developed a quantitative reverse transcriptase, polymerase chain reaction assay to map alpha 2 primary transcripts using primer pairs derived from different parts of the alpha 2 globin gene and its 3' flanking region. This approach has revealed the presence of steady state nuclear RNA past the poly(A) site of the alpha 2 globin gene at approximately 40% of the level of unspliced intron transcript. Furthermore, these 3' flanking transcripts diminish 500 bp into the 3' flanking region, identifying this part of the alpha 2 globin gene as the principal region of termination of transcription.


Screening for mutations by RNA single-strand conformation polymorphism (rSSCP): comparison with DNA-SSCP.

Single-strand conformation polymorphism (SSCP) is a simple method for detecting the presence of mutations in a segment of DNA, but the fraction of all mutations detected is unclear. We have evaluated SSCP for the detection of single-base mutations in the factor IX gene. Multiple conditions were examined including electrophoresis temperature, electrophoresis buffer concentration, acrylamide to bis-acrylamide ratio, and water-cooled versus fan-cooled gel apparatuses. Depending on conditions, 10-11 of 12 known mutations were detected in a 183 bp segment whereas only 11-14 of 22 known mutations were detected in a 307 bp segment. We hypothesized that single stranded RNA should have a larger repertoire of secondary structure because shorter hairpins form stable duplexes and the 2' hydroxyl group is available for sugar-base and sugar-sugar hydrogen bonds. By incorporating phage promoter sequences into PCR primers, RNA-SSCP (rSSCP) could be compared directly with standard DNA SSCP. rSSCP was generally superior to SSCP, especially for the 307 bp segment. In addition, the abundance of transcript produced as a result of rSSCP allows the rapid, nonradioactive detection of mutations by staining the gel with ethidium bromide. To gauge the utility of the method in a prospective manner, a blinded study was performed in which SSCP, rSSCP, and direct genomic sequencing were compared in 28 patients with hemophilia B. A total of 2.6 kb of factor IX genomic sequence was examined in nine regions ranging from 180 to 497 nucleotides of factor IX sequence. Sequence changes at 20 different sites were detected by direct genomic sequencing; 70% of these were detected by rSSCP while only 35% were detected by SSCP.


In vitro chromatin assembly promoted by the Xenopus laevis S-150 cell-free extract is enhanced by treatment with RNase A.

Cell-free extracts employed as chromatin assembly systems contain a myriad of proteins, polyanions and nucleic acids. The roles of ATP, MgCl2 and other cofactors in the catalysis of nucleosome formation by the Xenopus laevis oocyte S-150 have yet to be established unequivocally. In this study we examine the influence of RNA in the assembly process. Under reaction conditions that inhibit nucleosome formation (+ EDTA), pretreatment of the extract with RNase A revives the chromatin assembly machinery while the rate of DNA supercoiling is stimulated significantly. Addition of purified RNA blocks DNA supercoiling. Taken together, these data suggest that the parameters surrounding in vitro chromatin assembly are variable and subject to modulation by endogenous factors.


Cytokeratin immunoreactivity in malignant fibrous histiocytoma and spindle cell tumors: comparison between frozen and paraffin-embedded tissues.

Cytokeratin (CK) immunoreactivity in malignant fibrous histiocytoma (MFH) and other selected cases of spindle cell tumors were assessed using two cytokeratin monoclonal antibodies, AE1/AE3 and CAM 5.2. Frozen tissue was used to minimize the effects of fixation on keratin antigenicity; in addition, one block of fixed, paraffin-embedded tissue was tested for comparison. CK immunoreactivity was noted in nine frozen tissue samples (7/20 [35%] MFH, 1/3 schwannomas, 1/3 leiomyosarcomas). In the majority of cases, only rare individual positive cells were seen. Of 19 MFH cases in paraffin-embedded tissue, CK immunoreactivity was noted in three (16%). All 32 cases examined showed vimentin immunoreactivity. MFH must be added to the growing list of mesenchymal tumors exhibiting sporadic CK immunoreactivity. Such reactivity is less frequent in paraffin-embedded tissues. This finding has important implications for tumor diagnosis, particularly in the differential diagnosis of pseudosarcomatous carcinoma. Caution is recommended in the interpretation of CK immunoreactivity, particularly as it relates to speculations regarding histogenesis.


Peripheral neuroepithelioma: a light microscopic, immunocytochemical, and ultrastructural study.

Forty-two cases of peripheral neuroepithelioma (PN) retrieved from the files of the National Cancer Institute (Bethesda, MD) and the Pathology Department of Padua University, Italy, were reviewed. No sex predilection was observed (25M/17F) and ages ranged from 7 to 54 yr (median 22 yr). Roughly a third of the tumors were thoracopulmonary ("Askin tumor"), a third were axial, and a third were in extremities. A lobular pattern with rosettes or pseudo-rosettes characterized PN. Seventeen cases showed a strong diastase-sensitive PAS positivity. Transitional areas with an Ewing's-like appearance and, in one case, transition to malignant nerve sheath tumor have been documented. The presence of neuron specific enolase (NSE), S-100 protein, HNK-1, neurofilaments, vimentin, keratin (AE1-AE3), beta 2-microglobulin, chromogranin A, and synaptophysin was investigated using the avidin-biotin technique. Immunocytochemically, NSE (95% of cases), beta 2-microglobulin (77.5%), synaptophysin (73.3%), and S-100 protein (67.5%) were the most consistently positive markers. Ultrastructurally, PN is characterized by a primitive appearance, although it was routinely possible to recognize neural features such as primitive neuritic extensions and dense core granules, either in the cytoplasm or in the cellular processes. In our experience, a light microscopic picture of a primitive round cell tumor with a lobular pattern, and particularly with rosettes when present, with NSE and beta 2-microglobulin positivity by immunocytochemistry, ideally with positive synaptophysin, along with supportive electron microscopy, is required for the diagnosis of PN. Conversely, no one feature alone is generally sufficient for diagnosis, but does allow distinction from extraosseous Ewing's, which (like osseous Ewing's) lacks features of neural differentiation.


Estrogen and progesterone receptor detection in archival formalin-fixed, paraffin-embedded tissue from breast carcinoma: a comparison of immunohistochemistry with the dextran-coated charcoal assay.

The steroid receptor (estrogen (ER) and progesterone receptor (PR] status was studied in 94 cases of invasive breast carcinoma from three separate institutions. All cases had fresh tissue examined for ER and PR by the dextran-coated charcoal cytosolic assay (DCC), and each case was examined immunohistochemically for ER and PR from archival formalin-fixed, paraffin-embedded tissue. Immunohistochemical assays (IH) were reviewed blinded to the DCC results and scored in a semiquantitative fashion prior to comparison to the DCC results. Overall, there was agreement between DCC and IH in 89% of ER and in 87% of PR assays. Some 50% of the ER discorrelations were of the IH-positive DCC-negative type, while 27% of the PR discorrelations were of this type. In four cases, both ER and PR did not correlate between IH and DCC determinations, with two being IH (ER and PR) positive and DCC negative, and two of the opposite type. The results of the study show that steroid receptor assays performed on routinely processed formalin-fixed archival material are reliable and closely recapitulate the results of traditional biochemical assays. Results suggest that, in the cases where IH is positive while DCC is negative, the IH result may actually provide a more reliable receptor status of the tumor than does the DCC result. Semiquantitation of fixed tissue IH assays shows a trend toward quantitative correlation with DCC results, but this correlation is weak, and factors concerning fixation and processing are most likely to be responsible.(ABSTRACT TRUNCATED AT 250 WORDS)


Complete folding of bovine pancreatic trypsin inhibitor with only a single disulfide bond.

In the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) at neutral pH, only two one-disulfide intermediates accumulate to a significant extent, namely [5-55] and [30-51]. In this paper we describe a recombinant model of [5-55], designated [5-55]Ala, which was made by replacing the cysteine residues not involved in the disulfide bond with alanine. As judged by two-dimensional NMR, [5-55]Ala folds into essentially the same conformation as native BPTI. Moreover, like native BPTI, [5-55]Ala inhibits trypsin stoichiometrically. Thus, the disulfide-bonded intermediate [5-55] corresponds not to a partially folded protein folding intermediate but rather to an essentially completely folded protein. This conclusion provides an explanation for many of the thermodynamic and kinetic properties of [5-55] in the folding pathway of BPTI.


Intrinsic anion channel activity of the recombinant first nucleotide binding fold domain of the cystic fibrosis transmembrane regulator protein.

The first nucleotide binding fold (NBF-1) from the cystic fibrosis transmembrane regulator (CFTR) has been expressed in bacteria and found to bind ATP and to express anion channel activity when reconstituted onto a planar lipid bilayer. This evidence suggests that the NBF forms the anion-selective portion of the CFTR channel. We also found that the recombinant NBF-1 anion channel is blocked by ATP (1 mM), under which condition it appears to have a minimal conductance of approximately 9 pS and an ohmic current-voltage relationship. We further found that the recombinant NBF-1 bearing the delta F508 mutation has nearly identical anion channel activity to that of the wild-type protein but can be distinguished from wild type under bianionic conditions with chloride and gluconate. We conclude from these data that the anion channel activity of the recombinant NBF-1 could represent all or part of the anion conductance mechanism of CFTR and that the role of the ATP binding by the NBF could be to modulate this anion channel activity.


Role of the alpha v beta 3 integrin in human melanoma cell invasion.

The human melanoma cell line A375M expresses the vitronectin receptor (alpha v beta 3 integrin) on its cell surface. Treatment of A375M cells with either polyclonal or monoclonal anti-alpha v beta 3 antibodies resulted in stimulation of invasion through basement membrane matrices in vitro. Similar treatment of these cells with a monoclonal anti-alpha v antibody, which does not inhibit the adhesive function of the alpha v beta 3 antigen, also stimulated invasion; however, anti-beta 3 antibody treatment had no effect. Furthermore, pretreatment of the cells with vitronectin or addition of vitronectin to the basement membrane matrix also resulted in stimulation of invasion. Similar treatments with fibronectin receptor antibody or fibronectin had no effect on invasion. Analysis of type IV collagenase expression in cells treated with anti-alpha v beta 3 antibody showed higher levels of both the secreted 72-kDa enzyme and its mRNA. Signal transduction through the alpha v beta 3 integrin could underlie the elevated expression of metalloproteinase and the enhanced invasion of A375M cells through basement membrane matrices.


Primary cultures of endothelial cells from the human liver sinusoid are permissive for human immunodeficiency virus type 1.

Human endothelial cells isolated from hepatic sinusoids were infected in vitro with human immunodeficiency virus type 1 (HIV-1). An early sign of infection occurring in the culture was the formation of multinucleated cells. By double-labeling immunofluorescence, 5-15% of the cells recognized as endothelial cells owing to the presence of von Willebrand factor were found to contain HIV p24 and gp120 antigens after 2 weeks. Reverse transcriptase activity was released into the medium, and different steps in the process of viral budding were observed by electron microscopy. The virus produced by the endothelial cells was found to be infectious for CEM cells, a human T-cell line. CD4 molecules are present at the surface of the endothelial cells, as demonstrated by immunogold-silver staining and backscattered electron imaging. Treatment with an anti-CD4 antibody abolished productive infection of the sinusoidal endothelial cells. The possibility that endothelial cells of the liver sinusoid are infected in vivo with HIV remains to be clearly shown.


p21ras activation via hemopoietin receptors and c-kit requires tyrosine kinase activity but not tyrosine phosphorylation of p21ras GTPase-activating protein.

Products of the ras gene family, termed p21ras, are GTP-binding proteins that have been implicated in signal transduction via receptors encoding tyrosine kinase domains. Recent findings have defined a superfamily of hemopoietin receptors that includes receptors for a number of interleukins and colony-stimulating factors. The intracellular portions of these receptors show only restricted homologies, have no tyrosine kinase domain, and provide no clues to the mode of signal transduction. However, in most cases the factors stimulate tyrosine phosphorylation. We demonstrate here that ligand-induced activation of the interleukin (IL)-2, IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptors resulted in activation of p21ras in various hemopoietic cell lines. The only cytokine tested that binds to a hemopoietin receptor and that did not activate p21ras was IL-4. Activation of p21ras was also observed in response to Steel factor, which stimulates the endogenous tyrosine kinase activity of the c-kit receptor, as well as with phorbol esters, which activate protein kinase C. Experiments with protein kinase inhibitors implicated tyrosine kinase activity, but not protein kinase C activity, as the upstream signal in p21ras activation via these growth factor receptors. Attempts to demonstrate tyrosine phosphorylation of the p21ras GTPase-activating protein (GAP) were negative, suggesting that phosphorylation of GAP may not be the major mechanism for regulation of p21ras activity by tyrosine kinases.


Regulation of Cl- channels in normal and cystic fibrosis airway epithelial cells by extracellular ATP.

The rate of Cl- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl- conductance decreases the capability to secrete Cl-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl- secretion and apical membrane Cl- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl- conductance by external ATP is preserved in cystic fibrosis airway epithelia.


A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini.

In this study we have employed a synthetic peptide of the rab3a effector domain, rab3AL, to examine whether a rab-like low molecular weight GTP-binding protein is involved in protein release from the rat pancreatic acinar cell. The peptide was found to be a potent stimulator of amylase release from streptolysin-O-permeabilized pancreatic acini, with an EC50 of approximately 60 microM. Stimulation of amylase discharge by rab3AL did not occur using either intact acini or permeabilized acini depleted of ATP. In contrast, a different effector domain peptide of the rab2 protein, rab2AL, a peptide with distinct sequence homology to rab3AL, was unable to stimulate amylase release, suggesting the specificity of the rab3AL response to rab3-like proteins. rab3AL stimulated release at [Ca2+] that were nonstimulatory in the absence of the peptide (10 nM). rab3AL potentiated the effect of guanosine 5'-[gamma-thio]triphosphate on amylase secretion and decreased the amount of guanosine 5'-[gamma-thio]triphosphate required for maximal secretion, suggesting that these two agents interact to modulate a distal step(s) of secretion. The above results provide functional evidence for the role of a rab-like low molecular weight GTP-binding protein and its effector protein(s) in the control of protein release from pancreatic acini. Because the discharge response to rab3AL is near the maximal obtainable from permeabilized acini, our results would suggest that rab3-like proteins control an important step in regulated secretion of amylase.


Role of the KIT protooncogene in normal and malignant human hematopoiesis.

The role of the KIT protooncogene in human hematopoiesis is uncertain. Therefore, we examined KIT mRNA expression in normal human bone marrow mononuclear cells (MNC) and used antisense oligodeoxynucleotides (oligomers) to disrupt KIT function. KIT mRNA was detected with certainty only in growth factor-stimulated MNC. Expression was essentially abrogated by making MNC quiescent or by inhibiting myb gene function. Oligomers blocked KIT mRNA expression in a dose-response and sequence-specific manner, thereby allowing functional examination of the KIT receptor. In experiments with either partially purified or CD34(+)-enriched MNC, neither granulocyte nor megakaryocyte colony formation was inhibited by oligomer exposure. In contrast, KIT antisense oligomers inhibited interleukin 3/erythropoietin-driven erythroid colony formation approximately 70% and "stem cell factor"/erythropoietin-driven colony formation 100%. The presence of erythroid progenitor cell subsets with differential requirements for KIT function is therefore suggested. Growth of hematopoietic colonies from chronic myeloid leukemia and polycythemia vera patients was also inhibited, while acute leukemia colony growth appeared less sensitive to KIT deprivation. These results suggest that KIT plays a predominant role in normal erythropoiesis but may be important in regulating some types of malignant hematopoietic cell growth as well. They also suggest that KIT expression is linked to cell metabolic activity and that its expression may be regulated by or coregulated with MYB.


Modulation of dihydropyridine-sensitive calcium channels in heart cells by fish oil fatty acids.

The highly unsaturated n-3 fatty acids from fish oils, eicosapentaenoic acid [EPA; C20:5 (n-3)] and docosahexanoic acid [DHA; C22:6 (n-3)], prevent the toxicity of high concentrations of the cardiac glycoside ouabain to isolated neonatal rat cardiac myocytes. Arachidonic acid [C20:4 (n-6)] lacks such protective action. The protective effect of the n-3 fatty acids is associated with their ability to prevent high levels of cytosolic free calcium from occurring in response to the ouabain. This in turn results, at least in part, from a 30% reduction in calcium influx rate induced by the n-3 fatty acids. This protective effect is simulated by nitrendipine, a dihydropyridine inhibitor of the L-type calcium channels in cardiac myocytes. Nitrendipine (0.1 mM) alone, however, inhibits myocyte contractility, as do verapamil (10 microM) and diltiazem (1.0 microM). EPA or DHA (5 microM) blocks the inhibitory effects of nitrendipine but not those of verapamil or diltiazem. Bay K8644, a known dihydropyridine agonist of L-type calcium channels, produces a ouabain-like effect that is also prevented by EPA or DHA. Specific binding of [3H]nitrendipine to intact myocytes is noncompetitively inhibited by EPA or DHA in a manner that reduces the number of high- and low-affinity binding sites (Bmax) and increases their affinities. The fish oil fatty acids prevent calcium overload from ouabain and Bay K8644. They also prevent a calcium-depleted state in the myocytes caused by the L-type calcium channel blocker nitrendipine. The protective effects of the n-3 fatty acids appear to result from their modulatory effects on nitrendipine-sensitive L-type calcium channels.


Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes.

Interleukin 10 (IL-10), originally identified as a TH2 helper T-cell product able to inhibit cytokine production by TH1 cells, is highly homologous to BCRF1 (viral IL-10), an open reading frame in the Epstein-Barr virus genome. Here, we show that human and viral IL-10 stimulate DNA replication of B lymphocytes activated either via their antigen receptor or via their CD40 antigen. IL-4 and IL-10 display additive effects and induce a strong increase in the number of viable cells. Moreover, IL-10 induces activated B cells to secrete large amounts of IgG, IgA, and IgM, and the combination of IL-10 and IL-4 results in the secretion of the four immunoglobulin isotypes. Thus, IL-10 may play an important role in the amplification of humoral responses.


Insulin-like growth factor I gene expression is induced in astrocytes during experimental demyelination.

To investigate insulin-like growth factor I (IGF-I) and IGF-I receptor gene expression during experimental demyelination and myelin regeneration, young mice were fed cuprizone (( bis(cyclohexanone) oxaldihydrazone )). This copper-chelating agent produces demyelination in the corpus callosum and superior cerebellar peduncles, and when treatment is stopped, there is rapid remyelination. At intervals during cuprizone treatment and recovery, brain sections were hybridized with specific probes and immunostained with antibodies to determine the localization and relative amounts of IGF-I and IGF-I receptor mRNAs and peptides. In untreated littermates, IGF-I and IGF-I receptor mRNAs and peptides were not detected in white matter. In cuprizone-treated mice, high levels of both IGF-I mRNA and peptide were expressed by astrocytes in areas of myelin breakdown. Astrocyte IGF-I expression decreased rapidly during recovery and oligodendroglial expression of myelin-related genes increased. In severely demyelinated areas, immature oligodendroglia exhibited a transient increase in IGF-I receptor mRNA and peptide immunoreactivity during early recovery. This highly specific pattern of IGF-I induction in astrocytes during demyelination and the expression of the IGF-I receptor in regenerating oligodendrocytes during recovery suggest that IGF-I functions in the regulation of oligodendrocyte and myelin metabolism in vivo.


Fifth mutation in human immunodeficiency virus type 1 reverse transcriptase contributes to the development of high-level resistance to zidovudine.

It is recognized that high-level resistance to 3'-azido-3'-deoxythymidine (AZT, zidovudine, or Retrovir) is conferred by the presence of four mutations in the human immunodeficiency virus (HIV) reverse transcriptase [RT; deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (RNA-directed), EC 2.7.7.49] coding sequence. However, a number of clinical isolates have been observed that exhibit high-level resistance but contain only three of the four identified mutations (Asn-67, Arg-70, and Tyr-215). Construction of a molecular clone with this genotype gave rise to only a partially resistant virus, raising the possibility that an additional mutation existed in some clinical isolates. Using an HIV marker rescue system, we have mapped and identified a fifth mutation conferring resistance to zidovudine, namely, methionine to leucine at codon 41 of HIV RT. An infectious molecular clone containing this mutation together with three previously identified mutations in the RT coding sequence yielded highly resistant HIV after transfection of T cells. Direct detection of the fifth mutation in DNA samples from cocultured peripheral blood lymphocytes by the PCR revealed that it occurred relatively early in the development of zidovudine resistance. However, this mutation was only detected after the appearance of the codon 215 change in the RT coding sequence. Identification of this mutation in addition to the other known mutations conferring resistance enables rapid and direct correlation between an RT genotype and sensitivity of the virus.


Hoechst 33258 photosensitization of 5-iododeoxyuridine-substituted DNA to 365 nm light: dependence of dehalogenation on the dye-to-nucleotide ratio.

DNA containing 5-iododeoxyuridine substituted at levels ranging from 1 to 24% was irradiated at 365 nm in the presence of various amounts of the Hoechst dye 33258. The subsequent loss of iodine was measured by following the loss of radioactive iodine (125I) from filter immobilized DNA subjected to repeated washings. A plot of the photochemical cross section (sigma B) for this process as a function of r, the molar ratio of dye added per nucleotide present, shows a maximum at r = 0.04 to 0.06, followed by a decrease with increasing dye-to-phosphate ratios to give a minimum at r = 0.5. Increasing the amounts of dye further results in sigma B reaching a second maximum at r greater than or equal to 1, after which it levels off. An attempt was made to interpret these results in terms of the binding models currently employed for analyzing binding of Hoechst dye to DNA. Chain breaks as measured in alkali were also measured and found to be enhanced by the dye; the ratio of breaks per iodine loss was 0.9.


Inhibitory effect of prostaglandin delta 12-PGJ2 on cell proliferation and alpha-fetoprotein expression in HuH-7 human hepatoma cells.

9-deoxy-delta 9,delta 12-13,14-dihydro-prostaglandin D2 (delta 12-PGJ2) is a potent inhibitor of proliferation of tumor cells. In the present study, the effect of delta 12-PGJ2 on the alpha-fetoprotein(AFP) and the albumin gene expression was analyzed in HuH-7 human hepatoma cells. delta 12-PGJ2 inhibited the cell growth and reduced the medium AFP concentrations dose-dependently. To determine whether this decline of AFP depends only on the relative decrease in cell numbers by delta 12-PGJ2, or is in part, due to the decrease in the cellular AFP synthesis by delta 12-PGJ2, Northern blot analysis was performed in this study. By Northern blotting, it was shown that delta 12-PGJ2 caused a marked reduction in the levels of the AFP mRNA and the albumin mRNA. In contrast, the level of the beta-actin mRNA was not changed by delta 12-PGJ2. In the transient chloramphnicol acetyltransferase plasmid transfection experiments, delta 12-PGJ2 did not suppress the AFP enhancer activity, which possibly regulates both the AFP and the albumin gene expression in HuH-7 hepatoma cells, but resulted in the selective repression of the AFP and the albumin promoter activity. These results suggest that delta 12-PGJ2 suppresses not only cell growth but also expression of the AFP gene and the albumin gene at the transcriptional level in human hepatoma cells.


[The value of biopsy of the accessory labial salivary glands for the diagnosis of amylosis].

Diagnosis of amyloidosis depends on the demonstration of amyloid deposits in biopsies using specific stains. Recently, in addition to classical biopsies (kidney, liver, gum, skin, rectal mucosa), labial salivary gland biopsy has been recommended as safe diagnostic method. In our recruitment, it allowed the fortuitous discovery of amyloidosis in three patients suffering from rheumatoid polyarthritis or spondylarthritis. In five other patients (2 cases of familial amyloidosis, 1 dysglobulinemia, 2 primary cardiac amyloidosis), biopsy was performed for systematic search of amyloidosis. In five of these eight cases, a sicca syndrome was associated with the salivary deposits. These deposits were stained with congo red viewed in polarized light and with T thioflavine. Besides, Wright's method allowed to know the AL or AA type of amyloidosis and thus to guide the treatment. On the whole, labial salivary gland biopsy is a highly sensitive method for diagnosis of primary and secondary amyloidosis.


Formation of ion-permeable channels by tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF, cachectin), a protein secreted by activated macrophages, participates in inflammatory responses and in infectious and neoplastic disease states. The mechanisms by which TNF exerts cytotoxic, hormonal, and other specific effects are obscure. Structural studies of the TNF trimer have revealed a central pore-like region. Although several amino acid side chains appear to preclude an open channel, the ability of TNF to insert into lipid vesicles raised the possibility that opening might occur in a bilayer milieu. Acidification of TNF promoted conformational changes concordant with increased surface hydrophobicity and membrane insertion. Furthermore, TNF formed pH-dependent, voltage-dependent, ion-permeable channels in planar lipid bilayer membranes and increased the sodium permeability of human U937 histiocytic lymphoma cells. Thus, some of the physiological effects of TNF may be elicited through its intrinsic ion channel-forming activity.


Inhibition of development of Kaposi's sarcoma-related lesions by a bacterial cell wall complex.

In vitro and in vivo model systems for the study of human immunodeficiency virus (HIV)-associated Kaposi's sarcoma (KS) were used to evaluate compounds for their potential as therapeutic agents. A sulfated polysaccharide-peptidoglycan compound (SP-PG) produced by bacteria controlled the in vitro growth of acquired immunodeficiency syndrome (AIDS)-associated, KS-derived spindle-shaped cells (AIDS-KS cells) at noncytotoxic concentrations. Angiogenesis induced by AIDS-KS cells in the chicken chorioallantoic membrane assay was blocked by SP-PG, which also inhibited the vascular hyperpermeability response and the angiogenesis associated with the induction of KS-like lesions that develop after subcutaneous inoculation of AIDS-KS cells into nude mice. Suramin, pentosan polysulfate, and interferon alpha, which are currently in use for therapy of KS, were either less effective than SP-PG or much more cytotoxic, or both.


Using metaphors in therapy: dichos and Latino clients.

The clinical literature has given increased attention to the importance of culturally appropriate interventions with diverse populations. The use of metaphor as a tool in psychotherapy has become increasingly salient. Metaphor can also be used in work with Latino clients through the incorporation of dichos, or sayings, that exist in Mexican American and other Latino cultures. These folk sayings exhibit cultural beliefs and ideals embedded in figurative language that describe the human condition. The Spanish language has crystallized human situations, frailties, and anecdotes in the dichos used in everyday conversations. These dichos offer the clinician culturally viable tools for mitigating resistance, enhancing motivation, or reframing problems. Importantly, dichos provide an ambience that contributes to culturally sensitive treatment.


Photodynamic inactivation of retrovirus by benzoporphyrin derivative: a feline leukemia virus model.

The ability of a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), and either broad-spectrum (400-1200 nm) or narrow-band (600-700 nm) red light to kill feline leukemia virus (FeLV) and FeLV-infected cat T cells (cell line 3201) was investigated in culture medium containing fetal calf serum and in blood from infected cats. A molecular clone of FeLV, 61E, is minimally pathogenic and productively infects 3201 cells while causing no change in rate of cell division, viability, or size. Active virus (either free or within infected cells) was quantified by using a limiting dilution assay that involved cocultivation of test samples with naive 3201 cells, after which either the polymerase chain reaction or a reverse transcriptase assay was used to detect the presence of virus. It was shown that 61E-infected T cells in culture were slightly more sensitive to photodynamic killing than were uninfected cells. Infected cells and free virus were eliminated from whole blood taken from infected cats by using 4 micrograms per mL of BPD-MA and 40 J per cm2 of red light. These results correlate well with previous results with BPD-MA and vesicular stomatitis virus in whole human blood and suggest that this photosensitizer is a promising agent for the elimination of retroviruses that are either free or located within infected cells in blood.


Dramatic response of a metastatic carcinoid tumour to a combination of interferon and octreotide.

The combination of alpha-interferon and octreotide has rarely been tested in the treatment of carcinoid syndrome. We describe a patient who was moribund when treated with interferon alone, but enjoyed a dramatic response leading to disappearance of all symptoms, normalization of dU-5-HIAA, and restoration of his normal life-style when octreotide was initiated. Neither interferon nor octreotide could be withdrawn without reappearance of the symptoms, suggesting that the combination of alpha-interferon and octreotide may have synergistic effects in carcinoid syndrome.


Evaluation of heat sterilization of commercial rat diets for use in FDA toxicological studies.

Certified commercial rat diets, control and fortified, in the form of pellets and meal, were evaluated in a simulated subchronic rat feeding study. The diets were analyzed before and after autoclaving to determine nutrient integrity and loss, as well as the efficiency of autoclaving for removal of microbiological contaminants. Sterilization reduced the level of heat-labile vitamins, but protein level was minimally reduced. Sterilization eliminated most of the bacterial contaminants and virtually all the mold and yeast colonies. Male and female Osborne-Mendel rats (3-4 wk old) were fed control or sterilized diet for 6 wk. Both males and females consumed more pelleted chow than meal chow. This apparent difference in consumption may be due to wastage of pellets, because there were no differences in male or female growth during the 6-wk study. At necropsy, no gross pathology was noted, and organ weights did not differ significantly among the groups for either sex. Testicular weights were also similar among the groups. Blood serum proteins were analyzed by electrophoresis to screen for possible effects on various target organs. Gamma globulin levels for female rats fed sterilized meal were significantly reduced compared to levels for rats fed the control diet. These results suggest that either nutritional factors or heat inactivation of the microbes affects basal levels of humoral immunity, possibly by reduction of gut-mediated immune responses.


Refractile mycobacteria in Romanowsky-stained bone marrow smears. A comparison of acid-fast-stained tissue sections and Romanowsky-stained smears.

The appearance of mycobacteria was studied in Wright-stained bone marrow preparations of human immunodeficiency virus-infected patients and compared with acid-fast-stained trephine biopsy sections and culture results. Mycobacterium avium complex in Romanowsky-stained preparations may be seen as extracellular and intracellular clear or red refractile beaded rods and nonrefractile "negative images." Refractile mycobacteria were seen in 17 of 20 culture-positive cases. Acid-fast stain of the trephine biopsy demonstrated organisms in only 11 of the 20 cases. Thus, six cases were culture positive and contained refractile rods but had no acid-fast organisms on the trephine biopsy. No false-positive results were seen with Romanowsky stain; the three false-negative results for refractility also were negative with acid-fast stain. Examination of Romanowsky-stained smears or imprints for refractile mycobacteria provides a reliable and sensitive method to identify mycobacteria in this population. Romanowsky-stained bone marrow aspirate and imprint smears should be examined for refractile bacilli when mycobacterial infection is suspected.


Subtyping lymphocytes in peripheral blood by direct immunoalkaline phosphatase labeling and light scatter/absorption flow cytometric analysis.

Evaluation of blood cells to determine immunologic status is becoming an important clinical application of flow cytometric analysis. For a wider use of immunophenotyping technology in clinical laboratories, the authors developed a rapid method to detect monoclonal antibody-labeled cells using forward light scatter/absorption clinical flow cytometers such as the Technicon H*1 and Technicon H*2 differential complete blood count analyzers. Calf-intestinal alkaline phosphatase was conjugated to mouse monoclonal antibodies (anti-CD2, CD3, CD4, CD8, CD19) for direct immunoenzymatic labeling. The combination of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue-tetrazolium salt in diethanolamine buffer at pH 9.6 was selected as buffer/substrate to yield stable, insoluble, and very intense purplish-blue precipitates on the surface of the cells labeled with monoclonal antibody-alkaline phosphatase conjugates. Endogenous alkaline phosphatase in granulocytes was inhibited with levamisole. Early mild fixation of the white cells permitted incubation at 38 +/- 1 degrees C, which accelerated each step of the reaction without disrupting the cells throughout the procedure. The method is competitive with the direct immunofluorescence whole-blood method used on fluorescence flow cytometers in speed, sensitivity, and accuracy, as demonstrated with alkaline phosphatase-conjugated anti-CD2, CD3, CD4, CD8, CD19 monoclonal antibodies.


Immunohistologic demonstration of myocardial proteins with applications.

The authors used antibodies specific for creatine kinase MB isoenzyme (CK-MB) and myosin light chain-1 (MLC-1) for immunohistologic staining. At appropriate dilutions of antibody, frozen sections of human heart muscle were positive for both CK-MB and MLC-1, whereas sections of human skeletal muscle were negative for both proteins. Staining for both CK-MB and MLC-1 also was demonstrated in an immature teratoma. Furthermore, staining was localized to the rhabdomyosarcomatous elements within the teratoma; other components of the tumor did not stain for CK-MB or MLC-1. Biopsies of skeletal muscle revealed that regenerative, but not intact normal or degenerating, fibers also contained CK-MB and MLC-1. Immunohistologic stains for CK-MB and MLC-1 may be useful as tumor markers and as markers for regenerative muscle fibers.


Keratin-positive reticulum cells in fine needle aspirates and touch imprints of hyperplastic lymph nodes. A possible pitfall in the immunocytochemical diagnosis of metastatic carcinoma.

Fine needle aspirates and touch imprints of 36 hyperplastic (reactive) lymph nodes were tested for the presence of keratin and desmin. Keratin-positive cells with morphologic characteristics corresponding to extrafollicular (fibroblastic) reticulum cells were found in 18% of the fine needle aspirates and 42% of the touch imprints. The number of keratin-positive reticulum cells varied from 1 to greater than 30 per slide. Desmin-positive cells with similar morphology were found in 23% of fine needle aspirates and 37% of touch imprints, and the number of such cells per slide ranged from 2 to greater than 70. The relatively frequent occurrence of keratin-positive reticulum cells in these preparations from hyperplastic lymph nodes should be taken into account if keratin antibodies are used to search for carcinoma micrometastases.


Steroid hormone receptors in endometrial stromal sarcomas. A biochemical and immunohistochemical study.

Seven cases of endometrial stromal sarcoma (five low grade and two high grade) were analyzed immunohistochemically for the presence of estrogen and progesterone receptors. In four cases (three low grade and one high grade), these results were compared to biochemical findings. All low-grade endometrial stromal sarcomas were positive for progesterone receptors using immunohistochemical techniques. These results correlated well with biochemical evaluation of progesterone receptors. The high-grade endometrial stromal sarcomas were negative for progesterone and estrogen receptors by both methods. The advantages of immunohistochemical evaluation of steroid receptors have been well established in breast and endometrial carcinomas. This study demonstrates the usefulness of this technique in endometrial stromal sarcomas.


Somatostatin-producing neuroendocrine tumor of the ampulla (ampullary somatostatinoma). Evidence of prosomatostatin production.

Two cases of somatostatin-producing ampullary neuroendocrine tumors (somatostatinoma) are reported. The authors have characterized their immunoreactivity using antibodies specific for the amino- and carboxyl-terminal portions of prosomatostatin, the precursor of somatostatin in the normal synthetic pathway. Cytoplasmic staining was found using each of these two antibodies in the tumor cells of both ampullary somatostatinomas as well as in the cytoplasm of cells in the hypothalamus, crypt cells of the duodenal mucosa, mucosal cells of the biliary tract, D cells of the pancreatic islets, and parafollicular cells of fetal thyroid. These studies suggest that the synthesis of somatostatin in ampullary somatostatinomas occurs through the normal pathway from the precursor prosomatostatin.


Surgical excision of subfoveal neovascular membranes in age-related macular degeneration.

We studied the results of surgical excision of ten consecutive subfoveal choroidal neovascular membranes in ten patients with age-related macular degeneration. The criteria for surgical eligibility included the following: (1) a clearly identifiable subfoveal membrane occupying the entire foveal avascular zone, (2) a visual acuity of 20/200 or worse, (3) minimal subretinal hemorrhage, and (4) an associated exudative macular detachment. Six of the ten patients showed visual improvement at one-month and three-month follow-up visits and seven showed visual improvement by the six-month examination. All ten maculae remained attached without recurrence of subfoveal neovascular membranes throughout the follow-up period. These results suggested that surgical excision is a viable alternative to laser photocoagulation in patients with subfoveal neovascularization in age-related macular degeneration.


Zinc iodide-osmium tetroxide impregnation of the "tubulo-vesicular system" in Tomes' process of the rat incisor ameloblast.

Zinc iodide-osmium tetroxide (ZIO) is a nonspecific but selective impregnation method that visualizes a tubulo-vesicular system in cells. The detailed structure and three-dimensional distribution of this ZIO-impregnated system was studied in the Tomes' process of secretory ameloblasts in the rat incisor. The ZIO-impregnated system consisted of an extensive array of smooth membrane-bound thick and thin tubules and vesicles. The interconnected thick and thin tubules formed a complex "core network" in the central cytoplasm of Tomes' process that enmeshed and often surrounded individual secretory granules. From the core network, radial branches extended toward the smooth cell membrane of the interdigitating portion of Tomes' process. Although the core network and branches frequently appeared connected to the secretory granules and the cell membrane, stereo-pair electron microscopy failed to show conclusive evidence of such continuity. However, many coated vesiclelike structures were attached to the core network and its branches. No special relationship was found between interrod and rod secretory sites and the tubulo-vesicular network. In thick sections, the ZIO-impregnated tubulo-vesicular network occupied a considerable volume of cytoplasm. The vinblastine-labile nature of this network as demonstrated previously (Nanci et al., 1987) indicated that the system undergoes rapid and extensive turnover. Considering the dynamic nature and sheer volume of the tubulo-vesicular system, we propose that it be regarded as a major cell organelle.


Contrasting effects of alpha, beta, and gamma interferons on nonspecific suppressor function in multiple sclerosis.

Interferons are biological molecules with antiviral, antiproliferative, and immunomodulatory actions. Interferon alpha (IFN-alpha) and -beta are potentially useful in the treatment of multiple sclerosis (MS). IFN-gamma, in contrast, increases the frequency of exacerbations of MS. In this study, we compared the effect of recombinant human IFN-alpha, -beta, and -gamma on suppressor function in patients with MS. Nonspecific suppressor cell function, measured in a concanavalin A suppressor assay, was significantly decreased in 16 patients with progressive MS (mean percent suppression +/- SEM, 14.4 +/- 5.5 in patients with MS, 33.5 +/- 4.8 in 16 normal subjects; p less than 0.001). Recombinant human IFN-beta augmented suppressor function in MS to 45.4 +/- 5.1% (p less than 0.001) and in control subjects to 56.8 +/- 3.8% (p less than 0.001). Similarly, recombinant human IFN-alpha improved suppression in MS to 43.0 +/- 5.6% (p less than 0.001) and in control subjects to 51.1 +/- 5.9% (p less than 0.001). In contrast, recombinant human IFN-gamma had no effect on suppressor function in patients with MS and in control subjects. This study shows that IFN-alpha and -beta augment deficient suppressor function in MS, whereas IFN-gamma has no effect on suppressor function in the progressive phase of the disease.


Cyclic AMP metabolism in fragile X syndrome.

Cyclic AMP (cAMP) metabolism was studied in platelets from a series of 14 patients with fragile X syndrome (fra X) and 21 control individuals. 1-Isobutyl-3-methylxanthine was used to inhibit phosphodiesterase and thus measure cAMP production, prostaglandin E1 was used to assess receptor-mediated cAMP accumulation, and forskolin was used to directly stimulate the catalytic subunit. In patients with fra X, basal production was 63% of that of control subjects (p = 0.019). Prostaglandin E1- and forskolin-stimulated production were 61% (p = 0.039) and 56% (p = 0.012) of that of control subjects, respectively. cAMP production in 8 patients with fra X overlapped the control range, whereas measures of production in 6 patients formed a cluster with values lower than any of the 21 control subjects assayed, suggesting possible biochemical heterogeneity within patients with fra X. Results obtained from the group of patients with fra X suggest possible abnormal function or regulation of the catalytic subunit of adenylate cyclase in at least a subgroup of patients with fra X. Variability of biochemical findings in patients with fra X may reflect the known high variability of the clinical syndrome.


Regenerating and denervated human muscle fibers and satellite cells express neural cell adhesion molecule recognized by monoclonal antibodies to natural killer cells.

The monoclonal antibodies anti-Leu-19 and anti-NKH-1 recognize the CD56 differentiation antigen expressed on natural killer (NK) cells and on a T-cell subset. Because CD56 is an isoform of neural cell adhesion molecule (N-CAM), we examined its expression on human muscle using antibodies to Leu-19, NKH-1, and purified N-CAM in an immunohistochemical, immunoblot, and immunoprecipitation study on 70 muscle biopsy specimens from various muscle diseases and on human muscle in tissue culture. Anti-Leu-19, anti-NKH-1, and anti-N-CAM had identical immunoreactive patterns. In tissue sections, they specifically recognized the satellite cells and the regenerating or newly denervated muscle fibers; in tissue cultures, they immunoreacted with myoblasts and myotubes; and in the homogenates of myopathic muscle and cultured myotubes, they immunoprecipitated the same glycoprotein of 145- to 220-kd. The study concludes that (1) the commercially available monoclonal antibodies to NK cells, Leu-19 and NKH-1, are immunocytochemical markers for the satellite cells and the regenerating or newly denervated muscle fibers complementing conventional techniques in the diagnosis of patients with neuromuscular disorders; and (2) the CD56 is a common antigen shared by NK cells and muscle fibers during certain stages of muscle maturation, regeneration, or denervation. When expressed in the muscle, CD56 may facilitate the adhesion of cytotoxic lymphocytes to the muscle and play a role in muscle fiber injury.


[Supportive therapy in cancer treatment with colony stimulating factors and evaluation of their effect].

Several tumors are now curable. However, intensive chemotherapy or bone marrow transplantation is required for the cure, and thus myelosuppression and resultant serious infections are major obstacles for the cure of cancer. G-CSF and M-CSF are commercially available in Japan. By strict comparative studies, G-CSF has been shown to be a useful drug, shortening the recovery of neutrophils and reducing the incidence of documented infections. There have been no convincing data that G-CSF stimulates the regrowth of myeloid leukemia in vivo, and thus G-CSF is safe even in myeloid leukemia, if used with precaution.


[Detection of estrogen receptor (ER) mRNA by use of reverse transcriptase-polymerase chain reaction (RT-PCR) assay; comparison with dextran coated charcoal (DCC) assay and immunocytochemical assay].

A new method for estrogen receptor (ER) mRNA was performed on 33 human breast tumors, using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay by the method of Fuqua et al. In a preliminary experiment using the MCF-7 breast tumor cell line, ER/beta-actin ratio was almost same. ER protein was estimated by a dextran coated charcoal (DCC) assay and by an ER-immunocytochemical (ER-ICA) assay using a specific monoclonal antibody. We found RT-PCR assay correlates with ER-ICA assay (r = 0.664, p less than 0.01), whereas no significant correlation was seen between RT-PCR assay and DCC assay. These results suggests that RT-PCR assay is suitable for detection of ER from small amounts of tissue.


[A case of advanced ureteral squamous cell carcinoma showing complete response to combination chemotherapy with cisplatinum, vindesine, and bleomycin].

A 56-year-old Japanese female was diagnosed as advanced squamous cell carcinoma of the left ureter, stage IV (T4N0M0), and treated with anti-cancer drugs using cis-platinum, vindesine, and bleomycin. The tumor vanished after the treatment. The specimen after left total nephroureterectomy and hysterectomy showed only granulomatous change with necrosis and foreign giant cells but not cancer cells.


Controlled study of Pseudomonas cepacia and Pseudomonas maltophilia in cystic fibrosis.

In a retrospective study, children with cystic fibrosis who were colonised with Pseudomonas cepacia were compared with a control group who were colonised with Pseudomonas maltophilia. Out of 216 children with cystic fibrosis seen between 1983 and 1990, P cepacia was recovered from 13 (median age at colonisation 12.2 years) and P maltophilia from 23 (median age at first colonisation 6.1 years), and both organisms were recovered in five cases. With the exception of two patients with P cepacia in whom no other pathogens were found, all the patients with P cepacia or P maltophilia had co-colonisation with Pseudomonas aeruginosa. The lack of spread of P cepacia to siblings with cystic fibrosis, and the relative lack of inpatient contact between colonised and uncolonised patients suggest that cross infection is not the sole route whereby patients with cystic fibrosis become infected, but the possibility of cross infection cannot be excluded from our data. Three patients with P cepacia died, but two of these had shown appreciable respiratory deterioration before colonisation with P cepacia; there was no evidence of unexpected deterioration in the remainder or in the controls with P maltophilia. By 1990, the prevalence of P cepacia was 9/133 (7%) and that of P maltophilia was 13/133 (10%), but it was impossible to determine to what extent this increase was due to the introduction of the routine use of selective media. Further studies are required to establish whether patients with and without P cepacia should be segregated.


Acquired mutism: physiopathy and assessment.

The speechless patient presents a unique challenge to the clinician working with neurologically impaired adults. Acquired speechlessness, or mutism, has been associated with a variety of clinical states and syndromes after damage to central and peripheral nervous system structures. The intent of this paper is to summarize the reported states and syndromes associated with acquired mutism (eg, persistent vegetative state, akinesia), and to organize this information in a framework for clinical assessment of the speechless patient. For the purpose of discussion, speech production is divided into five interrelated processes: arousal; cognitive processing; affect and drive; motor initiation, planning, programming, and coordination; and execution of movement. Disorders characterized by mutism are classified according to the process or processes of speech production that primarily are affected. Each subtype of acquired mutism is characterized by a cluster of neurologic signs, which has been incorporated into a decision-making framework for use in a clinical setting.


Immunocytochemical labeling of cells in cortical vitreous from patients with premacular hole lesions.

We performed electron immunocytochemical staining for cytokeratins and glial fibrillary acidic protein on cortical vitreous obtained at the time of vitrectomy from two patients with premacular hole lesions. The specimens were thought to represent cortical vitreous because each consisted of a sheet of tissue that, in addition to being firmly attached around the foveal lesion, was attached around the disc and extended well into the periphery. The specimens contained a moderate number of cells and an abundant extracellular matrix. Most of the cells were found singly or in small clusters on the surface of the matrix. Preembedding immunostaining on one specimen showed several cells that stained for cytokeratins and several that stained for glial fibrillary acidic protein. The majority of the cells on the matrix appeared to express one of these two intermediate-filament proteins. Postembedding immunogold double labeling on both specimens showed that most cells were labeled for either glial fibrillary acidic protein or cytokeratins, but there were a few cells that unequivocally expressed both proteins simultaneously. These data suggest that the cortical vitreous of patients with some premacular hole lesions contains retinal pigment epithelial and glial cells that may contribute to abnormal vitreoretinal adherence and could play a role in macular hole formation.


3-amino-1,2,4-triazole inhibits macrophage NO synthase.

Murine macrophages activated by interferon-gamma and lipopolysaccharide become leishmanicidal through a process involving L-arginine-derived nitrogen oxidation products. Both nitrite secretion and parasite killing by activated macrophages were inhibited by 3-amino-1,2,4-triazole as well as the related compound, 3-amino-1,2,4-triazine. Moreover, NO synthase activity in cytosolic extracts of activated cells was inhibited by both compounds. 4-amino-1,2,4-triazole, an isomer of 3-amino-1,2,4-triazole, was without effect. Our results suggest that besides its known inhibitory effect on catalases and peroxidases, 3-amino-1,2,4-triazole is an inhibitor of NO synthase. The resemblance between the tautomeric form of 3-amino-1,2,4-triazole and the guanidino group of L-arginine, the natural substrate for NO synthase, might be responsible for the observed inhibition.


Cloning of murine and rat vascular cell adhesion molecule-1.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen 4 (VLA4). We have cloned the cDNAs for both murine and rat VCAM1 from endotoxin-treated lung libraries. Both sequences encode proteins with seven extracellular Ig-like domains, which show 75.9% and 76.9% identity, respectively, with human VCAM1. Both murine and human cell lines show VLA4-dependent binding to COS cells transiently expressing murine and rat VCAM1. Two mAbs, M-K/1 and M-K/2, which recognize an antigen on murine bone marrow stromal cell lines, bind to murine VCAM1 expressed in COS cells and block VCAM1-dependent adhesion, confirming that these mAbs recognize murine VCAM1.


Purification of nitric oxide synthase from bovine brain: immunological characterization and tissue distribution.

Nitric oxide (NO) synthase (EC 1.14.23) was purified to homogeneity from bovine cerebrum. The molecular weight of NO synthase was estimated to be 150 kDa by both SDS/PAGE and gel filtration at high salt concentration. For activity, the enzyme required NADPH, Ca2+, calmodulin and tetrahydrobiopterin as cofactors. Rabbit polyclonal antibody to bovine brain NO synthase reacted with 150 kDa NO synthase in various bovine and rat organs, including the brain, pituitary and adrenal glands, but not with that in stimulated macrophages, indicating that there are at least two immunologically distinct NO synthases.


Enhancement and alteration of bleomycin-catalyzed site-specific DNA cleavage by distamycin A and some minor groove binders.

The effects of compounds which bind in the DNA minor groove of A.T rich sequences, on bleomycin-catalyzed site-specific DNA cleavage were investigated by a DNA sequencing technique. Distamycin A enhanced bleomycin-catalyzed DNA cleavage in G.C rich sequences such as 5'-GGGGC-3' (under scoring; the cleaved nucleotide). The cleavage in such a sequence in the presence of distamycin A was greater than that in the absence of distamycin A by as much as about 100 times. Neither Hoechst 33258, 4',6-diamidino-2-phenylindole (DAPI) nor berenil caused extensive enhancement. The results suggest that the distamycin-induced conformational changes of DNA through interactions other than the DNA minor groove binding in A.T-rich sequences are specifically suitable for the bleomycin action.


Inhibition of IgE-mediated histamine release by myosin light chain kinase inhibitors.

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK) blocked IgE mediated histamine release from rat basophilic leukemia cell (RBL-2H3) and human basophils dose-dependently. Its IC50 was 20 nM for RBL-2H3 cells and 30 nM for human basophils. There was complete inhibition at the concentration of 1 microM. Wortmannin inhibited partially the A23187 induced histamine release from RBL-2H3 cells (40% inhibition at 1 microM). This inhibition was not accompanied by any significant effect on cytosolic free calcium concentration [( Ca2+]i). KT5926, another MLCK inhibitor, inhibited histamine release comparably with wortmannin and blocked to some degree the increase of [Ca2+]i in RBL-2H3 cells. Thus, the phosphorylation of myosin seems to be involved in signal transduction through Fc epsilon RI.


[Adjuvant and drug therapy of chronic pain in the head and neck area].

Head and neck pain caused of benign or malignant disease reduces remarkable the patient's quality of life. In the following are presented adjuvant and medicamentous methods for pain control. Surgery, irradiation and chemotherapy aim to diminish the tumor extension and reduce algesic transmitting substances in the periphery. Nerve blocs, cryoanalgesia and transcutaneous electrical nerve stimulation lead to an interruption of the painful spinal reflex arc. Active, passive and relaxation exercises prevent from dolorific muscular tensions. Psychological treatment, so as relaxation techniques in connection with behavior therapy, helps to develop coping strategies. The mainstay of pain relief is effective use of analgetics which should be given orally if possible, on a regular schedule and on an individualized basis according with the WHO guidelines.


A proliferation-related constraint on endogenous and interferon-induced 2-5A synthetase activity in normal and neoplastic Syrian hamster cells.

2-5A Synthetase is one of the most extensively characterized enzymes induced by interferon (IFN) and is the central enzyme in a pathway that may be involved in the control of cellular proliferation. We examined the activity of this enzyme in normal diploid Syrian hamster cells (FC13) and their neoplastically transformed derivatives (BP6T); the former cell strain possesses regulated proliferative control, while the latter cell line has escaped from this control. A significant threefold increase in 2-5A synthetase activity was observed in density-arrested versus proliferating FC13 cells, whereas endogenous enzyme activity was uniformly low in BP6T cultures. The increase in enzyme activity in FC13 cultures was not accompanied by the production of IFN at a detectable level, but was parallelled by an increase in the intracellular level of 2',5'-oligoadenylate. IFN treatment resulted in a differential induction of enzyme activity depending on the proliferative state of FC13 cells. After IFN treatment, BP6T cells and subconfluent FC13 cells responded similarly with a fivefold increase in enzyme activity, whereas confluent FC13 cells displayed only a 1.4-fold increase. 2-5A Synthetase enzyme activity reflected steady-state mRNA levels in BP6T and subconfluent FC13 cells. In contrast, a noncoordinate regulation of 2-5A synthetase mRNA expression and enzyme activity was detected in confluent FC13 cells, suggesting that posttranscriptional mechanisms may be involved. The different patterns of endogenous and IFN-induced 2-5A synthetase enzyme activity in FC13 and BP6T cells found in this comparative study may represent an alteration fundamental to the loss of proliferative control in transformed cells.


Immunological variation and immunohistochemical localization of HIV-1 Nef demonstrated with monoclonal antibodies.

To study the immunological and immunohistochemical nature of HIV-1 Nef. Monoclonal anti-Nef antibodies were generated and used to identify antigenic epitopes in Nef, to study immunological cross-reactivity between Nef from different isolates and to reveal the subcellular localization of Nef. Monoclonal antibodies against recombinant HIV-1 Nef protein (BRU isolate) were generated in BALB/c mice. The epitope mapping was carried out with the use of overlapping 15-20mer lipopeptides linked to a lipid group at the amino-terminus. Immunoperoxidase method was used for histochemical studies. Ten stable antibody-producing clones, mainly of the immunoglobulin (Ig) G1 subtype, with strong Western blot and enzyme-linked immunosorbent assay reactivity toward the recombinant Nef protein, were obtained. The epitopes recognized were located on amino-acid sequences 21-41, 31-50, 51-71, 61-80, 151-170, 161-180, and 171-190. All 10 monoclonal antibodies also reacted with the native Nef of HIV-1BRU, and eight reacted with native HIV-1IIIB. Most antibodies also reacted with Nef from more divergent HIV-1 strains. In Western blotting, two forms of Nef (24 and 27 kDa) were observed with most isolates studied. Immunohistochemical staining of HIV-1-infected H9 or MT-4 lymphoid cells demonstrated that Nef was expressed mainly in the Golgi complex and at the nuclear membrane, but occasionally also in the nucleus. The nuclear localization of Nef was especially frequent in the HIV-1-infected MT-4 cells. Our findings suggest that Nef is expressed in two isomorphic forms, and that it may also act as a nuclear protein and thus have a direct regulatory function at the RNA/DNA level.


HIV-induced, HIV-specific in vitro antibody response by B-cells from HIV-seropositive individuals.

Recent studies have shown that B-cells from HIV-infected patients can secrete anti-HIV antibodies in vitro and that they represent 20-40% of immunoglobulin (Ig)-secreting B-cells in vivo. This study was designed to investigate the precise role of HIV in this in vitro antibody production. B-cells from HIV-infected patients [asymptomatic, n = 28; symptomatic (AIDS), n = 14], from seronegative adult volunteers (n = 22) and subjects at high risk for HIV infection (n = 15) were cultured in vitro in the presence of pokeweed mitogen, Staphylococcus aureus cowan or HIV, and T-cells or interleukins (IL). Non-specific Ig production and specific anti-HIV antibody (Ab) production were measured by enzyme-linked immunosorbent and Western blot assays. We found that HIV induced a specific response in cultured B-cells from seropositive patients, in contrast with cultured B-cells from uninfected normal individuals. The characteristics of the HIV-induced response differed from those of a spontaneous or a mitogen-induced response. Anti-HIV Ab production was optimal on day 8-10, when B-cells were cultured with recombinant IL-2 and recombinant interferon-alpha in the presence of infectious virus or recombinant gp160 Env protein. The anti-HIV Ab were mainly directed against Env proteins. Interaction of HIV with B-cells involved surface IgG but not CD4 antigen. Autologous CD8+ T-cells had a non-specific inhibitory effect. Both CD5+ and CD5- B-cells produced anti-HIV Ab. No anti-HIV Ab production was observed in B-cells from high-risk HIV-seronegative individuals. HIV (infectious virus or gp160) can induce B-cells from infected patients to secrete specific anti-HIV Ab in vitro.


Oligonucleotide therapeutics.

Recent studies have focused on the ability of oligonucleotides to affect the genetic processes of many organisms, including viruses. Hence, oligonucleotides may represent a future source of biotechnologically derived compounds of therapeutic importance for diseases such as cancer and AIDS.


Short-term memory and verbal learning with auditory phonological coding defect: a neuropsychological case study.

A patient is described with a rarely reported linguistic syndrome: he could repeat words but not nonwords. The patient produced semantic paraphasias in repetition and could read both words and nonwords flawlessly. His basic difficulties were localized in auditory phonological coding, identifying a clinical picture called "phonemic deafness." Short-term memory and verbal learning results suggested that a standard, selective short-term memory defect can be induced by auditory phonological coding deficits as well as by "pure" short-term memory capacity limitation and other phonological deficits. Findings also provided evidence that lexical-semantic code can allow normal verbal learning.


Interaction between secretin and cholecystokinin-octapeptide in the exocrine rat pancreas in vivo and in vitro.

This study investigates the interaction between physiological doses of the synthetic gut hormones, cholecystokinin-octapeptide (CCK8) and secretin on pancreatic juice secretion in the anaesthetized rat and on amylase secretion and Ca2+ and Mg2+ mobilization in isolated pancreatic segments and acinar cells. CCK8 (150 pmol kg-1 h-1) and secretin (100 pmol kg-1 h-1) evoked marked time course increases in pancreatic juice flow, total protein output and amylase secretion in the anaesthetized rat when administered separately compared to saline controls. Simultaneous intravenous infusion of CCK8 and secretin did not yield either an additive response or a potentiation but instead it caused a decrease in secretory responses. Administration of either polymyxin B (10(-8) mol kg-1 h-1) or staurosporine (10(-8) mol kg-1 h-1), two protein kinase C inhibitors, simultaneously with both CCK8 and secretin caused a further decrease in all secretory parameters. Superfusing pancreatic segments with either CCK8 (10(-11) M) or secretin (10(-11) M) elevated amylase output compared to the smaller response with a combination of CCK8 and secretin. Combining staurosporine (10(-6) M) with CCK8 and secretin resulted in a further decrease in amylase output. CCK8 (10(-11) M) evoked a large increase in radiolabelled Ca2+ influx into pancreatic segments and elevated cytosolic free Ca2+ concentration ([Ca2+]i) in acinar cells loaded with the fluorescent dye, Fura-2. Secretin (10(-11) M) alone had no significant effect on Ca2+ mobilization but it markedly attenuated the increases in radiolabelled Ca2+ influx and [Ca2+]i elicited by CCK8. In superfused pancreatic segments CCK8 (10(-11) M) evoked a net efflux of Mg2+ whereas secretin (10(-11) M) induced a net uptake of Mg2+. Combining secretin with CCK8 also resulted in a net uptake of Mg2+. The results indicate that both Ca2+ and Mg2+ mobilization may be associated with the interaction between CCK8 and secretin in the rat pancreas.


Influence of 4 different membrane oxygenators on inflammation-like processes during extracorporeal circulation with pulsatile and non-pulsatile flow.

The influence of four different membrane oxygenators (HF 4000, BOS-CM 50, CML 2, Maxima) on leucocyte count, concentrations of PMN-elastase, clotting factor XII, AT-III, C1-INH, alpha 2-antiplasmin and C3a was registered before, during and after CPB with pulsatile and nonpulsatile flow in 80 male patients aged between 36 and 67 years. With all systems tested, there was a drop in the concentrations of clotting factor XII, AT-III, C1-INH and alpha 2-antiplasmin in the early extracorporeal circulation (ECC) phase, exceeding the average hematocrit reduction accounted for by dilution. This drop was the least distinct with CML 2 systems, both with pulsatile and nonpulsatile perfusion, indicating system-inherent influences. Leucocyte cound and PMN-elastase concentration rose significantly during ECC irrespective of oxygenator tested of flow type applied. The rise in leucocyte count even continued for about 4 h after ECC. During the first 40 min of ECC, these changes were paralleled by a significant rise in C3a concentration, suggesting complement activation as a main cause for PMN activation. However, there is reason to suppose involvement of further mechanisms operating in PMN activation, since the elevated C3a-concentrations began to fall off while leucocyte count and PMN-elastase concentrations were still increasing.


Mediastinal pancreatic pseudocyst.

Pseudocysts of the pancreas are a rare cause of a mediastinal mass. They are clinically characterized by the combination of thoracic symptoms (shortness of breath, dysphagia, pleural effusions) with complaints in the upper abdominal quadrants and weight loss. The diagnosis is usually made by CT scan or MRI including upper abdominal views. Internal drainage via an abdominal route performed either as cystogastrostomy or cystojejunostomy is the treatment of choice.


Characterization of apolipoprotein B mRNA editing from rabbit intestine.

Apolipoprotein (apo) B-48 is generated by a unique physiological process. Cytidine 6,666 of the apo B primary transcript is posttranscriptionally converted to a uridine by an RNA editing mechanism that transforms the codon for glutamine 2,153 to a termination codon. The editing reaction can be duplicated in a cell-free extract. In this study, the apo B-48 mRNA editing activity derived from partially purified extracts of rabbit enterocytes was characterized. The optimum conditions for the editing reaction were determined to be a salt concentration of 0.125-0.150 M NaCl or KCl, a pH of 8-8.5, and a temperature of 30 degrees C. The reaction rate was linear up to 45 minutes and was proportional to the editing extract concentration. No metal ion cofactors, DNA or RNA cofactors, or energy requirements were identified. At optimum conditions, the reaction followed Michaelis-Menten kinetics, with a Km of 0.4 nM for the rabbit RNA substrate. In addition, the reaction rate was enhanced by the addition of 25 micrograms/ml heparin or 40% glycerol. The characteristics of the editing reaction suggest that it is catalyzed by a nucleotide sequence-specific cytidine deaminase that is either a single enzyme or a multimeric protein.