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At least 55 records · Page 3Linked to original sources
PubMed1988

Novel assay for pancreatic cellular damage: 1. Characterization of protein profiles in human pancreatic cytosol and purification and characterization of a pancreatic specific protein.

The protein patterns of cytosols from normal human pancreas and pancreatic carcinoma were studied by a two-dimensional separation technique using high-performance liquid chromatography followed by isoelectric focusing on polyacrylamide gels and visualization of the focused proteins by Coomassie Blue staining. Almost identical protein patterns were obtained for 20 different specimens from normal pancreas, whereas quite different protein patterns were found in 12 samples of pancreatic carcinoma. A major protein in normal pancreatic cytosol, not identical to any macromolecule previously tested as a marker for pancreatic function, was selected for further studies. The protein was not found in specimens of pancreatic carcinoma. It was purified by a single step chromatofocusing procedure, focused at pH 6.9, and moved as one single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 44,500 daltons. Total amino acid analysis revealed a high concentration of glutamic acid, leucine, and lysine. The purified protein had no amylase activity or lipase immunoactivity. It constituted approximately 2% of the total normal pancreatic cytosol protein. Later immunological studies have shown the protein to be highly specific for normal human pancreas, indicating a possible future use as a marker for pancreatic cell damage.

Adenocarcinoma↗
PubMed1987

New methodology for the synthesis of 2'(3')-O-aminoacyl oligoribonucleotides related to the 3'-terminus of aa-tRNA.

A new synthetic approach to the title compounds is reported. It is based on the phosphotriester methodology and uses a unique combination of protecting groups (Fmoc and Ph for the aglycons; DMT, CPTr and Mthp for the carbohydrate hydroxy functions; 2-CIPh for the phosphodiester bonds; BPOC for amino acid). This enables selective deprotection of the blocked oligonucleotide intermediates in two steps (oximate and acid treatments) to yield the 2'(3')-O-aminoacyl oligoribonucleotides suitable for biochemical investigations.

Indicators and Reagents↗
PubMed1979

An immunological approach to cell separation of the central nervous system.

In this paper I review the various steps of development required for an immunological approach to cell separation. Key points include choice of brain region, isolation procedure, ligand selection, functional assays for separated cell population, and selection of separation procedure. I review briefly some results we have obtained using cerebellar cell populations isolated from 10-day-mice.

Animals↗
PubMed1992

Hematoencephalic barrier. Ultrastructure and histophysiology of the endothelium capillary of the neuronal nuclei of the mesencephalon.

The ultrastructure of the dorsal periaqueductal nucleus capillaries of the mesencephalon in the cat was studied under the electron microscope in relation to the hematoencephalic barrier, and its four structural levels: 1. Endothelium; 2. Basal membrane; 3, Pericytes; and 4. Glial prolongations. An analysis was performed of what occurs in these four components (in a non-experimental histophysiological state, and without manipulation by markers) in the thinnest capillaries of the centre of the mesencephalic neuronal nucleus. Special attention was placed on the first diffusion barrier formed by the endothelium capillary as the intimate guardian of the Central Nervous System (C.N.S.) neurons. The C.N.S. capillaries are formed from the continuous endothelium, with no fenestrations, and hermetic joining complexes, without pinocytosis vesicles on both sides of the plasmatic membrane (adluminal and external), and surrounded by a continuous basal membrane. The non-fenestrated capillaries of the C.N.S. are less permeable than those with similar characteristics located in other areas. In the C.N.S. these capillaries form a selective physiological barrier which determines the size of the molecules that are permitted to cross the capillary wall. It is suggested that the electron-dense globules found in the endothelium cytoplasm may be molecules assimilated from the blood, which might represent the first level or step to the selective diffusion entrusted to the hematoencephalic barrier. It is also suggested that the elongated electron-dense particles found in the endothelium cytoplasm and basal membrane may be macromolecules which are normally retained for an active defensive function. They would represent the first and second level or steps of the retention performed by the hematoencephalic barrier which blocks their passage to the confined space of the perivascular capillary.

Animals↗
PubMed1976

Role of proteolytic enzymes in biological regulation (a review).

Many enzymes, hormones, and other physiologically active proteins are synthesized as inactive precursors (zymogens) that are subsequently converted to the active form by the selective enzymatic cleavage (limited proteolysis) of peptide bonds. The ultimate agency of activating enzymatic function is limited proteolysis, either in a single activation step or in a consecutive series (cascade). The specificity of each activation reaction is determined by the complementarity of the zymogen substrate and the active site of the attacking protease. The sequence of consecutive activation reactions is regulated by the specificity of each enzyme, whereas the degree of amplification of the initial stimulus is determined by the efficiency of each activating step. Zymogen activation produces a prompt and irreversible response to a physiological stimulus, and is capable of initiating new physiological functions. Typical examples are the precesses of blood coagulation, fibrinolysis, complement activation, hormone production, metamorphosis, fertilazation, supra-molecular assembly, and digestion. The zymogens of the pancreatic serine proteases, in particular, have served as models for detailed studies of the nature of the molecular changes that are involved in the dramatic increase in enzymatic activity that ensues upon limited proteolysis of the zymogen.

Animals↗
PubMed1991

Compression of the ambulatory ECG by average beat subtraction and residual differencing.

We implemented a method for compression of the abulatory ECG that includes average beat subtraction and first differencing of residual data. Our previous investigations indicated that this method is superior to other compression methods with respect to data rate as a function mean-squared-error distortion. Based on previous results we selected a sample rate of 100 samples per second and a quantization step size of 35 microV. These selections allow storage of 24 h of two-channel ECG data in 4 Mbytes of memory with a minimum rms distortion. For this sample rate and quantization level, we show that estimation of beat location and quantizer location can significantly affect compression performance. Improved compression resulted when beats were located with a temporal resolution of 5 ms and coarse quantization was performed in the compression loop. For the 24-h MIT/BIH arrhythmia database our compression algorithm coded a single-channel of ECG data with an average data rate of 174 bits per second.

Algorithms↗
PubMed1986

Inositol phospholipid metabolism in the kidney.

The unique features of renal phosphoinositide metabolism include an increase in tissue phosphoinositide levels induced by PTH. The significance of this finding remains unclear. Another unusual finding is the localization of phospholipase C activity in a BBMV preparation. As suggested in the review, the transducing mechanism involving cleavage of phosphoinositides by a phospholipase C would be expected to include a close association between phospholipase C and the plasma membrane. However, few attempts to localize phospholipase C activity in the plasma membrane have succeeded. The kidney also plays an unusual role in inositol metabolism in that it is the only organ that significantly catabolizes inositol. The kidneys also synthesize inositol. There is an enormous concentration of inositol in the outer medulla. This coexistence of significant inositol synthesis, breakdown, and the presence of extremely high amounts of free inositol is an intriguing but unexplained phenomenon. The substantial rate of endogenous renal inositol synthesis does not, however, preclude inositol deficiency states. There is a deficiency of inositol in diabetic peripheral nerve and in glomeruli isolated from diabetic rats. Such deficiencies may arise from a disturbance in the balance of synthesis, breakdown, and excretion of inositol, and particularly from the competition of glucose with the inositol transporter in the proximal tubule. Future studies of renal phosphoinositide metabolism need to address both basic cell biological questions and broader physiological or functional questions. The more basic issues include the question of which phosphoinositide is being attacked by agonist-stimulated phospholipase C. That is, are all the events explained by hydrolysis of PtdIns(4,5)P2, or are the other phosphoinositides hydrolyzed as well? Also, it would appear that stimulated phosphoinositide metabolism occurs quite early following receptor occupation, but there is still no way of selectively blocking stimulated phosphoinositide metabolism to see if it is a necessary first step in a cascade of events leading to cell response. Thus, the relationship of stimulated phosphoinositide metabolism to cell functions remains incompletely understood. At least two cellular functional or biochemical changes associated with stimulated phosphoinositide metabolism in the kidney have been identified, prostaglandin production and mesangial cell contraction. The regulation of prostaglandin production and its relationship to stimulated phosphoinositide metabolism are subjects of continuing study. The topic was recently reviewed by Hassid.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗
PubMed1990

Ipsilateral and cross-over elongation of the motor nerve by nerve grafting: an experimental study in sheep.

In difficult reconstructions, ipsilateral or cross-over nerve grafting is sometimes necessary to achieve reinnervation and motor function. This experimental study in sheep was to answer the question of limitation of elongation of a motor nerve by grafting, the question of the optimal time for suturing the nerve graft to the muscle nerve, and the question of the successful application of this surgical technique in extremities. In 18 sheep, the vastus nerve was elongated by a saphenous nerve graft as long as possible up to 30 cm (step 1). In 10 animals the nerve graft was applied ipsilaterally, and in 8 animals it was used as a cross-over nerve graft to the contralateral limb. The time between nerve grafting and connection of the distal end of the nerve graft to the freshly cut rectus nerve supplying the rectus femoris muscle (step 2) was variable: 0, 3, 6, 9, and 12 months. In all animals, the final experiments (step 3) were performed 6 months after the last operation (step 2). Muscle force measurements in the rectus femoris muscle and quantitative analysis of the number and diameter of myelinated nerve fibers in cross sections of the nerve biopsies at different levels showed that elongation of a motor nerve by nerve grafting is principally not limited. The functional results were rather inhomogeneous and therefore unpredictable (ipsilateral group: maximum tetanic tension = 27 to 172 N; cross-over group: 0 to 227.5 N). Nevertheless, crossover nerve grafting is recommended for selected cases even in extremities. There was no correlation between the time interval between the two operations and the functional or morphologic results, although better functional results were obtained when the distal nerve suture (step 2) was performed some months after nerve grafting (step 1). A clear correlation was found only between the number of regenerated axons in the rectus nerve behind the second suture line and the muscle function.

Animals↗
PubMed1988

Functional role of specific secreted and cell surface molecules in tumour cell invasion and metastasis.

The invasion and metastatic spread of tumour cells are complex biological processes involving an array of cell surface and secreted protein molecules which function as specific recognition and catalytic agents in each step of the malignant cascade. In order to begin to identify these molecules and implicate them as true functioning agents, our laboratory has developed selected panels of monoclonal antibodies that have been screened for their ability to inhibit specific manifestations of the malignant phenotype in various model systems. Since monoclonal antibodies recognize only single and unique determinants, it is hypothesized that any monoclonal antibody which is specifically antifunctional will allow for the identification and isolation of relevant protein molecules.

Animals↗
PubMed1977

The representation of membrane admittance.

An integral representation for the membrane admittance in terms of its known current response to a voltage step function is presented. It is demonstrated that the frequency-dependent terms in the contribution to the membrane admittance by the ion-selective conductance of the nerve membrane are proportional to the static conductances. The additional information contained in the real and imaginary parts of the membrane admittance should allow the parameters of the ion conductance to be determined. Eventually, these measurements should also give information about the electric dipole displacement currents of the conductance systems themselves, and about the metabolically supported active ion transport currents that maintain the ion concentration gradients.

Animals↗
PubMed1976

Radiologic evaluation of the function of the cervical spine.

Cervical functional examinations initiated in 1965 suggested the need of a correlated, multifactorial expression of the cervical sagittal movement function, allowing comparative evaluations of physiologic-diagnostic significance. Technical aspects of the successive steps of investigation, together with the final morphologic-functional formula are described. The significance of the normal and abnormal functional patterns, identified in a selected series of 300 cases, is finally discussed.

Adult↗
PubMed1992

[Improvement of clinical results by digitalization of a single channel cochlear implant].

For 1973 we have been among the first to claim the multichannel cochlear implant superiority regarding single channel efficacy. However we actually thing that single channel cochlear implant is indispensable in case of total ossified cochlea, and very useful when efficacy/coast ratio must be considered. In order to narrow the gap between multi and single channel device efficacy, we used the new microprocessors possibilities to digitalize the analogic emietter of the single channel system, which we designed in 1987. Owing to a PC keyboard the new emietter allows the speech therapist to select the frequency band width of the input signal, and to determine the threshold level and the dynamic value as a function of six steps values of the stimulus wave frequency. These improvements supply the patient with a better speech intelligibility excepted for vowels discrimination. Comparison of clinical results obtained through the two analogic and digital systems are reported on 4 post lingually and 4 pre lingually deaf patients.

Adolescent↗
PubMed1992

Cytotoxicity and antitumor activity of carzelesin, a prodrug cyclopropylpyrroloindole analogue.

The cyclopropylpyrroloindole analogues are DNA minor-groove binders containing a cyclopropyl group, which mediates N3-adenine covalent adduct formation in a sequence-selective fashion. Carzelesin (U-80244) is a cyclopropylpyrroloindole prodrug containing a relatively nonreactive chloromethyl precursor to the cyclopropyl function. Activation of carzelesin requires two steps, (a) hydrolysis of a phenylurethane substituent to form U-76073, followed by (b) ring closure to form the cyclopropyl-containing DNA-reactive U-76074. The formation of the DNA-reactive U-76074, via U-76073, from carzelesin was shown to proceed very slowly in phosphate-buffered saline (t1/2 greater than 24 h) but to occur rapidly in plasma from mouse, rat, dog, and human (initial t1/2 values ranging from 18 min for mouse to 52 min for rat) and in cell culture medium (t1/2 approximately 40 min). Although carzelesin was less potent in terms of in vitro cytotoxicity and in vivo optimal dosage and showed low affinity for binding to DNA, it was therapeutically more efficacious against mouse L1210 leukemia than was U-76074 or adozelesin (U-73975), another cyclopropylpyrroloindole analogue which is currently in phase I clinical trials. Carzelesin also proved to be more efficacious than U-76074 or adozelesin against mouse pancreatic ductal 02 adenocarcinoma, a system reported to be resistant to every agent tested. Carzelesin was highly effective against this tumor and produced 97% tumor growth inhibition. In addition, i.v. administered carzelesin showed significant activity (National Cancer Institute criteria) against i.v. or s.c. implanted Lewis lung carcinoma, i.p. or s.c. implanted B16 melanoma, s.c. implanted colon 38 carcinoma, and five s.c. implanted human tumor xenografts, including clear cell Caki-1 carcinoma, colon CX-1 adenocarcinoma, lung LX-1 tumor, ovarian 2780 carcinoma, and prostatic DU-145 carcinoma. Carzelesin treatment produced 100% complete remissions (no palpable tumor mass at the termination of the experiment) in mice bearing early-stage human ovarian 2780. Pharmacologically, carzelesin proved to be relatively schedule and route independent and was highly active against i.p. implanted L1210 leukemia, regardless of whether the analogue was given i.v., i.p., s.c., or p.o. These results, collectively, suggest that carzelesin is absorbed and distributed well. Both carzelesin and adozelesin caused marked tumor shrinkage in mice bearing human lung LX-1 or advanced-stage human ovarian 2780 carcinoma; however, tumor regrowth occurred shortly after the treatment with adozelesin was stopped. Little or no apparent tumor regrowth occurred after treatment with carzelesin.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenocarcinoma↗
PubMed1992

Use of the HPRT gene and the HAT selection technique in DNA-mediated transformation of mammalian cells: first steps toward developing hybridoma techniques and gene therapy.

In 1956, I decided to apply my experience in microbial genetics to developing analogous systems for human cell lines, including the selection of mutants with either a loss or gain of a biochemical function. For instance, mutants resistant to azahypoxanthine showed a loss of the HPRT enzyme (hypoxanthine phosphoribosyl transferase), whereas gain of the same enzyme was accomplished by blocking de novo purine biosynthesis with aminopterin, while supplying hypoxanthine and thymine (HAT selection). Using HAT selection, we: (i) genetically transformed HPRT- mutant cells to HPRT+ wild type by using DNA extracted from HPRT+ cells, and (ii) selected HPRT+ hybrid cells by fusing HPRT- D98/AH2 cells with skin cells. These approaches, which we dubbed in 1962 as a 'first step toward gene therapy', contributed to the later development of (i) cell fusion techniques, (ii) the development of monoclonal antibodies, (iii) routine transformation of mammalian cells with cloned genes, and (iv) methods for creating transgenic organisms.

Animals↗
PubMed1987

Amplification and expression of foreign genes in cells producing polyoma virus large T-antigen.

Polyoma virus (Py) large T-Antigen (LT) can promote the amplification of viral genomes integrated in the chromosomal DNA of rat fibroblasts, and this phenomenon requires the interaction of the LT protein with the viral origin of DNA replication. To compare the rate and the modality of selectable amplification events promoted by the Py LT with cellular-driven events, we constructed a double expression vector containing a murine dihydrofolate reductase (dhfr) cDNA and the bacterial chloramphenicol acetyl transferase (cat) gene controlled by the viral regulatory region. The plasmid was introduced into a rat cell line constitutively producing a temperature sensitive LT (cl 2), and clones were selected in low concentration of methotrexate (MTX). Three cl 2 transformants and one control cell line lacking LT were propagated at temperatures permissive (33 degrees C) and non permissive (39 degrees C) for LT function and, subsequently, challenged in one step with high MTX dosage at 39 degrees C. While the control line produced the same number of colonies irrespective of the temperature of propagation, the three LT positive lines yielded between 2 and greater than 100 times more colonies following propagation at permissive temperature, indicating that the presence of Py LT considerably increased the rate of amplification of integrated sequences linked to the viral origin of replication. In all cases the amplification event involved the exogenous and not the endogenous dhfr gene, and overexpression of the cat gene occurred as a result of co-amplification with the selectable dhfr sequences. Analysis of the structure of the amplified domain in the various resistant derivatives revealed that, in the presence of the viral protein, amplification occurred within the boundaries of the primary plasmid insert. In the absence of a functional LT protein, amplification both internal or involving adjacent host DNA were observed.

Animals↗
PubMed1987

Phosphorylation and dephosphorylation of soluble proteins in human eosinophils.

The effect of phorbol 12-myristate 13-acetate (PMA), calcium ionophore (A23187), opsonized zymosan (OZ), and N-formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe) on protein phosphorylation was examined in purified eosinophils (eos) isolated from human peripheral blood. Eos were prelabeled with [32P]orthophosphate, stimulated with several activating agents for varying periods of time. The soluble proteins were then analyzed by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. In resting eos, there was phosphorylation of endogenous soluble proteins with molecular weights of 12, 16, 21, 40, and 66 kilodaltons (kDa). PMA, a potent activator of oxidative metabolism, induced phosphorylation of 19-, 40-, and 67-kDa proteins. A23187, a strong degranulating stimulus, caused phosphorylation of 40-, 53-, and 67-kDa proteins. OZ, a relatively weak stimulus for eos function, caused phosphorylation of 30-34-, 59-, 67-, and 93-kDa proteins. In addition, all the above stimuli caused a time-dependent dephosphorylation of 21-kDa protein. In contrast, f-Met-Leu-Phe caused neither phosphorylation of new proteins nor dephosphorylation of preexisting eos proteins. These findings demonstrate that selected stimuli affect phosphorylation of soluble eos protein. These results also suggest that phosphorylation of specific proteins in eos is an intermediary step in external stimulus-induced cell activation, which may involve many different cell functions.

Blood Proteins↗
PubMed1985

Functional recognition of bacterial mitogens by B lymphoma cells: reactivity of WEHI 279.1 to lipopolysaccharide and selection of nonreactive variants.

To analyze functional mitogen recognition by reactive B lymphocytes, we studied the effects of bacterial lipopolysaccharide (LPS) on the growth of the WEHI 279.1 B lymphoma line (W279). We found that LPS inhibits, in a dose-dependent manner, the growth of W279 cells in culture and that it reduces the frequency of cells growing as clones under limiting dilution conditions. Furthermore, we show that differential reactivity of "wild-type" cells to increasing LPS concentrations reflects the heterogeneity in the lymphoma cell population and the frequencies of "resistant" variants to each mitogenic concentration. This allowed us to derive variant tumor cell lines and clones, no longer LPS sensitive, either from mass cultures or, in a single-step selection, under limiting dilution conditions in the presence of low and high concentrations of LPS. Although mitogen reactivity is progressively lost upon prolonged culture, resistance to LPS was found to be a stable trait in selected variants, suggesting that it results from loss of functional mitogen recognition by the reactive cells. The specificity of mitogen reactivity or resistance was shown by the fact that some of the variant clones are still reactive to T helper cell-derived factors and others are not. Thus reactivity to LPS and to T cell factors can be separated, suggesting that the cell lines described here provide new tools for the biochemical analysis of B cell activation.

Animals↗
PubMed1992

The yeast WBP1 is essential for oligosaccharyl transferase activity in vivo and in vitro.

Asparagine-linked N-glycosylation is a highly conserved and functionally important modification of proteins in eukaryotic cells. The central step in this process is a cotranslational transfer of lipid-linked core oligosaccharides to selected Asn-X-Ser/Thr-sequences of nascent polypeptide chains, catalysed by the enzyme N-oligosaccharyl transferase. In this report we show that the essential yeast protein WBP1 (te Heesen et al., 1991) is required for N-oligosaccharyl transferase in vivo and in vitro. Depletion of WBP1 correlates with a defect in transferring core oligosaccharides to carboxypeptidase Y and proteinase A in vivo. In addition, in vitro N-glycosylation of the acceptor peptide Tyr-Asn-Leu-Thr-Ser-Val using microsomal membranes from WBP1 depleted cells is reduced as compared with membranes from wild-type cells. We propose that WBP1 is an essential component of the oligosaccharyl transferase in yeast.

Alleles↗