Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization.

Several of the endothelial cell polypeptide mitogens that have been described probably play a role in blood vessel homeostasis. Two overlapping complementary DNA clones encoding human endothelial cell growth factor (ECGF) were isolated from a human brain stem complementary DNA library. Southern blot analysis suggested that there is a single copy of the ECGF gene and that it maps to human chromosome 5 at bands 5q31.3 to 33.2 A 4.8-kilobase messenger RNA was present in human brain stem messenger RNA. The complete amino acid sequence of human ECGF was deduced from the nucleic acid sequence of these clones; it encompasses all the well-characterized acidic endothelial cell polypeptide mitogens described by several laboratories. The ECGF-encoding open reading frame is flanked by translation stop codons and provides no signal peptide or internal hydrophobic domain for the secretion of ECGF. This property is shared by human interleukin-1, which is approximately 30 percent homologous to ECGF.

Isolation and sequencing of a cDNA clone homologous to the v-sis oncogene from human endothelial cells.

A clone containing the 3' end of the mRNA for the human c-sis gene (homologous to the B chain of platelet-derived growth factor) was isolated from a cDNA library derived from human umbilical vein endothelial cells and then sequenced. The analysis of possible translation products in all three reading frames indicated that the A chain of platelet-derived growth factor was not coded for within the 3' end of the c-sis mRNA. The 3' end of the mRNA for c-sis is contained in or adjacent to exon 6.

Cloning and expression of the variable regions of mouse myeloma protein MOPC315 in E. coli: recovery of active FV fragments.

Recombinant DNA techniques were used to clone and express the FV portion of MOPC315, a mouse myeloma protein with a high affinity for 2,4-dinitrophenyl (DNP). The FV fragment consists of a heterodimer of heavy and light chain variable domains (VH and VL). Two separate bacterial plasmid constructs, containing either a variable region cDNA for the light chain or a variable region synthetic gene for the heavy chain demonstrated high levels of expression (150-200 mg/L) under control of the bacteriophage T7 promoter. Recombinant chains were initially recovered as inclusion bodies and then dissolved separately in 8 M urea, combined together, and refolded by subsequent chaotrope removal. Biologically active FV was affinity purified from the chain mixture by specific binding to DNP-lysine-Sepharose. Yields of active material as high as 20% were obtained with activity confirmed by fluorescence quench analysis. The purified FV displayed a binding affinity of 4.8 +/- 0.3 x 10(-7) M which was identical to the native FV. Chimeric FVs composed of recombinant and native chain mixtures yielded similar results. Recombinant MOPC315 FV activity was also obtained using a single chain construct (sFV), in which recombinant VH and VL were linked via a (Gly4Ser)3 spacer region. Binding affinity of the sFV was shown to be the same as the recombinant and native FVs. The ease of purification and characterization of active MOPC315 as the recombinant and native FVs. The ease of purification and characterization of active MOPC315 FV makes this system useful in the study of the optimization of antibody production in bacteria.

Identification of novel peptide antagonists for von Willebrand factor binding to the platelet glycoprotein Ib receptor from a phage epitope library.

We have constructed a fusion phage epitope library in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 10(7) different epitope bearing phage, has been used in an attempt to identify inhibitors of the von Willebrand factor (vWF)-platelet Glycoprotein Ib interaction. The library was screened with a monoclonal antibody (RG46) that recognizes the GPIb binding domain of vWF (amino acids 445-733). A total of 30 clones falling into 8 classes have been identified that react with the RG46 antibody. Isolates from all 8 classes are positive by immunoblot analysis. The amino acid sequence of the gene III fusion protein from positive clones showed a strong homology to the known RG46 epitope. Peptides identified from the screen were synthesized and used to demonstrate that some of the synthetic peptides exhibited inhibitory activity towards ristocetin induced binding of vWF to the GPIb receptor. Thus, we have demonstrated that screening a fusion phage epitope library with a monoclonal antibody that inhibits vWF binding to the GPIb receptor can be a useful tool not only for mapping antibody recognizing determinants, but also can serve as a source for identifying novel peptides that are antagonists for vWF binding to the platelet GPIb receptor.

Convergence pattern of cortical and interposital influences on rubrospinal neurons of the cat.

In adult semi-chronically implanted sedated cats discharges of single rubrospinal (RS) neurons were tested for selective activation of movement-evoking foci within pericruciate cortex (CX; area 4) and anterior division of cerebellar interpositus nucleus (IN). It was found that a very high incidence of neuronal responses was obtained only when stimulating IN and CX foci which controlled the same joint with respect to that moved from activation of the rubral foci including the recorded neurons. In these cases very frequent convergence phenomena were observed by activating IN and/or CX foci. Analysis of response patterns showed that RS neurons included in a focus controlling a given muscle were excited by IN foci for the same muscle (agonist foci) and inhibited from IN foci for the antagonist muscle (antagonist foci). In contrast, the same RS neurons were inhibited by agonist CX foci and excited by antagonist CX foci. Such an organization suggests that the motor cortex plays a competitive role in modulating the action of the interposito-rubrospinal system.

Nerve root compression by herniated intradiscal gas. Case report.

Intervertebral disc degeneration of any etiology may be associated with the formation of spaces or clefts within the disc. Gas collects within these spaces and can be seen roentgenographically. A case is presented in which intradiscal gas herniated into a connective tissue capsule, displacing the left S-1 nerve root and producing symptoms and signs identical to those of a herniated nucleus pulposus. The pathophysiology of gas within a disc space and the possibility that it may herniate much like the nucleus pulposus is discussed.

Hyperthermia for the treatment of brain tumors.

Brain malignancy, either primary or metastatic, in general is associated with a very poor outcome in spite of the best therapy modern medicine has to offer. The multimodality approach appears to offer the best chance of achieving our goal of improved survival (quality and quantity) and ultimately cure. Hyperthermia, though its application to brain cancer remains experimental, has proven itself in its ability to improve cancer therapy results. There are many methods available to apply hyperthermia to the brain and its application, thus far, has been quite safe. Continued research in this exciting field is warranted.

Polyalkylphenyl-sulphonic acids with acid groups of variable strength from animal-vegetable wastes.

The insoluble organic fraction isolated from rice-hulls residues and animal fecal matter mixture is sulphonated in liquid SO3 at 200 degrees C to a water soluble sulphonate (III). III is compared to the sulphonate (IV) obtained from the same mix after composting. Both products have been found to contain mixtures of molecules with close molecular weight. These molecules consist of a central cicloaliphatic cluster with peripheral pending aromatic chains. III and IV appear to have the same sulphonation degree. However, the latter contains higher concentrations of cicloaliphatic fragments and of amide, phenol and ether bonds, but less carboxylic and amine functional groups. These differences may be reasonably traced back to the starting materials. By comparison with commercial lignosulphonates derived from the paper and pulp industry, the above arylsulphonates are likely candidates for a variety of applications in the chemical industry and in agriculture. We conclude that sulphonation, even under the drastic experimental conditions of this work, does not seem to erase the memory of the parent matter structure. This reaction is capable of upgrading recalcitrant organic matter in vegetable waste residues to an interesting variety of lignosulphonates.

Polyalkylphenyl-sulphonic acid with acid groups of variable strength from compost.

The insoluble organic fraction (humin-like material, HLM) from rice hull-dairy cattle compost is well converted into water soluble HLM-sulphonate by reaction in liquid SO3. Microanalytical, potentiometric, molecular weight, and NMR data are consistent with a highly homogeneous polymeric arylsulphonate having 4000 Da MW, 1.3 sulphonic groups per aromatic ring and significant content of carboxylic and phenolic groups. By comparison with structure-property relationships for commercial lignosulphonates derived from the pulp and paper industry, the above arylsulphonate is likely to be a candidate at a variety of applications in the chemical industry and in agriculture. Therefore, sulphonation is a means for upgrading composts HLM to the same uses as for commercial lignosulphonates.

Unilateral enhancement of early and late blink reflex components in hemidystonia.

The side-to-side differences in the EMG activity of the early and late components of blink reflex, regarded as revealing the state of excitability of the brain stem reflex centers, have been analyzed in patients with unilateral dystonia without demonstrable brain lesions. It has been observed that both early and late responses of direct blink reflex were higher on the side affected by hemidystonia than on the contralateral one, while the latency values were in the normal range. Possibility that an abnormal output from the striatum towards the brainstem structures involved in blink reflex appears on the affected side of hemidystonic patients is discussed.

Swallowing disorders: management data.

Aim of the investigation was to assess the workload and verify the results of oropharyngeal dysphagia management in a large state hospital by means of a descriptive, observational prospective study and descriptive statistical analysis. 81 patients [37 females, 44 males, mean age 61.3 (+/- 13) years] suffering from oropharyngeal dysphagia were evaluated and treated in the in- and outpatient Divisions of the "Azienda Ospedaliera S. Giovanni Battista" in Turin. Treatment of oropharyngeal dysphagia included changes in consistency and texture of food, compensatory postures of head, strengthening exercises for oropharyngeal muscles, and stimulation of pharyngeal sensitivity. In data collection and analysis, the following were used as outcome measures: mode of nutrition delivery (oral, enteral, parenteral), dietary adjustments, presence of aspiration or penetration, and use of compensatory head positioning. Results showed that the number of patients fed by parenteral or enteral tube (50/81 prior to treatment) dropped to 36/81 upon discharge from hospital. Those unable to take anything by mouth, from 55 dropped to 9. The number of patients with aspiration or penetration dropped, respectively, from 47 and 8 to 20 and 4. Postural changes were used in 15 cases. Data obtained indicate that oropharyngeal dysphagia rehabilitation outcomes are promising. Better understanding of the rheological characteristics of food and a stricter, more rigorous evaluation of the outcomes on activities and social participation are warranted.

Production and functional characterization of a recombinant fragment of von Willebrand factor (vWF): an antagonist to platelet receptor GP Ib.

We expressed a recombinant peptide fragment (Ser445-Val733) of human von Willebrand factor (vWF), containing the binding domain for the platelet receptor of GP Ib, in E. coli. This 33 kD peptide blocks binding of the intact vWF molecule to GP Ib in the presence of modulators. Thus, it offers potential as an antithrombotic agent. High level expression was achieved in a plasmid construct driven by the bacteriophage T7 promoter. The peptide was solubilized from inclusion bodies in strong chaotrope, then reduced and alkylated. Following purification, formulation at pH 3.5, and lyophilization, the reconstituted experimental product (RG 12986) exists as an equilibrium of monomer and dimer species. When formulated above pH 5.0, soluble aggregates are formed; these solutions have less bioactivity than RG 12986. Interestingly, the non-aggregated state of RG 12986 remains conserved following dilution and incubation with platelet-poor plasma. The overall purification/low pH formulation strategies may be applicable to other E. coli recombinant proteins having a tendency to aggregate following removal of chaotrope near physiologic pH when in a concentrated format.

Endoscopic management of sinonasal vascular tumors without embolization: Our experience with vessel ligation

Objective@#To present our surgical experience and technique in performing endoscopic sinus surgery for vascular sinonasal tumors without pre-operative embolization using intraoperative ligation of the external carotid artery or its distal branches.@*Methods@#Design: Retrospective Series. Setting: Tertiary Private Teaching Hospital. Participants: Seven Patients. @*Results@#Out of 7 patients (5 males, 2 females, aged 12 to 64 years old) with non-embolized vascular sinonasal tumors, 2 had juvenile angiofibroma, 3 had a benign vascular tumor (hemangiopericytoma, hemangioma and a vasoformative solitary fibrous tumor), and 2 had a malignancy (rhabdomyosarcoma, squamous cell carcinoma). Four (57.1%) had external carotid artery ligation, two (28.6%) had internal maxillary artery ligation and one (14.2%) had sphenopalatine artery ligation. The mean intraoperative blood loss was 2447.1 mL (range 900mL to 5,000mL) and average operation duration was 7.6 hours (range 2.9 hours to 14.5 hours). The average amount of transfused blood products was 1785.7mL (zero to 3,000mL). The average hospital stay was 7 days (range 2 to 13 days) with one post-operative complication (ICU admission for hypotension from intraoperative blood loss). @*Conclusion@#Intraoperative ligation of the ECA or its distal branches to disrupt the vascular supply of sinonasal tumors may provide a viable means of preventing excessive intraoperative blood loss in patients with non-embolized vascular sinonasal tumors.

Nucleotide sequence of rat alpha 1-acid glycoprotein messenger RNA.

The complete nucleotide sequence of rat alpha 1-acid glycoprotein (alpha 1-AGP) mRNA has been determined from cloned double-stranded cDNA. The coding portion of the mRNA was bounded at the ends by a 5'-untranslated region of 35 nucleotides in length and a 3'-untranslated region of 119 nucleotides in length. The 3'-untranslated region contains the characteristic AAUAAA sequence ending 18 nucleotides from the 3'-terminal poly(A) segment. The 5'-region of the mRNA contains two in-phase AUG codons separated by 12 nucleotides. Comparison with the known NH2-terminal amino acid sequence of serum rat alpha 1-AGP suggests that the primary translation product of the mRNA contains an additional 14 or 18 amino acids that are not present in the mature form of the protein, which contains 187 amino acids. The inferred amino acid sequence of rat alpha 1-AGP and the known amino acid sequence of human alpha 1-AGP have several regions of identity clustered in the NH2-terminal portion of the proteins. The carboxyl-terminal regions show significantly less homology. Six potential asparagine glycosylation sites are found in the rat sequence, and four of these sites are in positions similar to known glycosylation sites in the human protein. Furthermore, three of these potential glycosylation sites are in a region that exhibits extensive amino acid sequence conservation, suggesting that this region may be important for the biological function of alpha 1-AGP.

Cloning of rat alpha-fetoprotein 3'-terminal complementary deoxyribonucleic acid sequences and preparation of radioactively labeled hybridization probes from cloned deoxyribonucleic acid inserts.

Double-stranded complementary deoxyribonucleic acid (cDNA) was synthesized from rat yolk sac alpha-fetoprotein (AFP) mRNA, inserted into the PstI site of plasmid pBR322 by an oligo(deoxyguanylic acid).oligo(deoxycytidylic acid) joining technique, and cloned in Escherichia coli chi 1776. A plasmid containing an inserted AFP double-stranded cDNA with a contiguous poly(adenylic acid) [poly(A)] segment was identified and subsequently employed in a new method for preparing AFP-specific hybridization probe. Following an initial digestion of the AFP plasmid with HindIII to create an open, recessed 3' end, lambda exonuclease III was employed to remove the DNA strand opposite the coding strand of the cDNA insert. Oligo(thymidylic acid) was then annealed to the poly(A) segment and employed as primer for E. coli DNA polymerase I to synthesize a 32P-labeled cDNA copy of the AFP coding strand. The single-stranded cDNA product was easily isolated by sedimentation through isokinetic alkaline sucrose gradients. Hybridization with this AFP-specific cDNA probe showed that the yolk sac contained a 6-fold greater concentration of AFP mRNA than that of the fetal liver. AFP mRNA was also found in the normal adult liver, but at a much lower level than in the fetal liver. The concentrations of AFP mRNA in Morris hepatomas 7777 and 8994, however, were significantly elevated to a 2- to 3-fold higher concentration that in the fetal liver.

Simple rapid method for the synthesis of radioactively labeled cDNA hybridization probes utilizing bacteriophage M13mp7.

Double-stranded cDNA sequences for rat alpha 1-acid glycoprotein and rat glutathione S-transferase mRNAs were inserted into the Pst I site of bacteriophage M13mp7 and used to develop a new method for preparing specific cDNA hybridization probes directly from cloned template DNA. A palindrome sequence surrounding the Pst I site in the vector DNA permitted single-stranded DNA isolated from the recombinant phage to fold back, thus forming a stable hybrid bounded on the ends by a large loop of M13mp7 single-stranded DNA and a small loop of inserted foreign DNA. A primer corresponding to an internal sequence of the foreign DNA was hybridized, then Escherichia coli DNA polymerase I was used to synthesize a 32P-labeled complementary DNA copy of the cloned inserted DNA. The single-stranded cDNA reaction product was easily isolated by subsequent sedimentation through alkaline sucrose gradients. Gel electrophoresis of the labeled cDNA product, after denaturation with glyoxal, indicated a single discrete band with an electrophoretic mobility corresponding to the length of the inserted DNA sequence. About 95% of the cDNA product formed S1 nuclease-resistant hybrids in hybridization reactions with excess RNA in solution. DNA sequences complementary to the M13mp7 vector DNA were not detected in the cDNA product. Thus, these M13mp7-derived probes are the functional equivalent of cDNA copies to mRNAs and can be employed for quantitative measurements of mRNA concentration. This simple, rapid method probably can be used for most cloned DNA sequences to yield single-stranded radioactively labeled DNA, without contaminating vector DNA sequences, for virtually any hybridization requirement.

New aporphine alkaloids of Ocotea minarum.

Fourteen aporphine alkaloids were isolated from the leaves of a Brazilian Lauracea, Ocotea minarum Nees (Mez). The known alkaloids were identified through their physico-chemical properties as: leucoxylonine (VII), dicentrine (IV), ocoteine (V), leucoxine (VI), ocopodine (VIII), predicentrine (IX), dicentrinone (XIV) and thalicminine (XV). Six new aporphine alkaloids were also isolated: ocotominarine (I), ocominarine (III), nor-leucoxylonine (XI), iso-oconovine (xii), 4-hydroxydicentrine (XIII) and ocominarone (XVI). Their structures were determined using spectroscopic methods and chemical correlations.