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Biomedical subjects

J Walter

Publications and source records attributed to J Walter.

At least 19 recordsLinked to original sources
PubMed1992

BsuBI--an isospecific restriction and modification system of PstI: characterization of the BsuBI genes and enzymes.

The enzymes of the Bacillus subtilis BsuBI restriction/modification (R/M) system recognize the target sequence 5'CTGCAG. The genes of the BsuBI R/M system have been cloned and sequenced and their products have been characterized following overexpression and purification. The gene of the BsuBI DNA methyltransferase (M.BsuBI) consists of 1503 bp, encoding a protein of 501 amino acids with a calculated M(r) of 57.2 kD. The gene of the restriction endonuclease (R.BsuBI), comprising 948 bp, codes for a protein of 316 amino acids with a predicted M(r) of 36.2 kD. M.BsuBI modifies the adenine (A) residue of the BsuBI target site, thus representing the first A-N6-DNA methyltransferase identified in B. subtilis. Like R.PstI, R.BsuBI cleaves between the A residue and the 3' terminal G of the target site. Both enzymes of the BsuBI R/M system are, therefore, functionally identical with those of the PstI R/M system, encoded by the Gram negative species Providencia stuartii. This functional equivalence coincides with a pronounced similarity of the BsuBI/PstI DNA methyltransferases (41% amino acid identity) and restriction endonucleases (46% amino acid identity). Since the genes are also very similar (58% nucleotide identity), the BsuBI and PstI R/M systems apparently have a common evolutionary origin. In spite of the sequence conservation the gene organization is strikingly different in the two R/M systems. While the genes of the PstI R/M system are separated and transcribed divergently, the genes of the BsuBI R/M system are transcribed in the same direction, with the 3' end of the M gene overlapping the 5' end of the R gene by 17 bp.

Amino Acid Sequence↗
PubMed1992

Local growth of a Burkitt's lymphoma versus disseminated invasive growth of the autologous EBV-immortalized lymphoblastoid cells and their somatic cell hybrids in SCID mice.

Specific host-graft interactions, as well as intrinsic properties of transferred cell, determine tumorigenicity in xenogeneic systems. We compared the growth characteristics of human B-lymphoid cell lines in SCID mice with the well characterized growth pattern in nude mice and observed striking differences in malignancy in the respective hosts. Two cell lines derived from the same individual, the Epstein-Barr-virus(EBV)-positive Burkitt's lymphoma BL 60 (BL) and the autologous EBV-immortalized lymphoblastoid cell line IARC 277 (LCL) were used. In addition, we tested somatic cell hybrids (HYB) of both cell lines, which despite the LCL-like differentiation phenotype show the de-regulated c-myc expression pattern of the parental BL line, assumed to be a critical factor in BL pathogenesis. Subcutaneously (s.c.) injected BL cells produced local progressively growing tumor masses at the injection site without distant metastases in both nude and SCID mice. Although both mouse strains possess the same genetic background (BALB/c) and differ only in the B-cell sub-set, the growth patterns of the LCL and hybrids were completely different. In contrast to the regressive behaviour of LCL and hybrids in nude mice, these lines show invasive and disseminated progressive growth in SCID mice. Peripheral lymph nodes an thymic tissue were preferentially colonized, whereas mucosal-associated lymphoid tissue (Peyer's patches and appendix) and spleen were not infiltrated. The preferential migration of lymphocytes to certain tissues is termed homing in a syngeneic system and mediated by homing receptors and vascular addressins. The "homing" of LCL and hybrids into lymphoid SCID mouse tissue suggests a strong interaction with the endothelial cells of the host. Detailed phenotypic analysis of BL, LCL and 3 different hybrids was performed using an antibody panel against differentiation and adhesion markers. Overall dominance of the LCL phenotype was observed in the hybrids, as indicated by cytology, tumor growth, dissemination and the pattern of surface-marker expression. The c-myc activation in hybrids does not appear to influence growth behavior.

Animals↗
PubMed1992

High plasticity of multispecific DNA methyltransferases in the region carrying DNA target recognizing enzyme modules.

Multispecific cytosine C5 DNA methyltransferases (MTases) methylate more than one specific DNA target. This is due to the presence of several target recognizing domains (TRDs) in these enzymes. Such TRDs form part of a variable centre in the MTase primary sequence, which separates conserved enzyme core sequences responsible for general steps in the methylation reaction. By deleting, rearranging and exchanging several TRDs of multispecific MTases, we demonstrate their modular character; they mediate target recognition independent of a particular TRD or core sequence context. We show also that multispecific MTases can accommodate inert material of non-MTase origin within their variable region without losing their activity. The remarkable plasticity with respect to the material that can be integrated into this region suggests that the enzyme core sequences preceding or following it form separable functional domains. In spite of the documented flexibility multispecific MTases could not be endowed with novel specificities by integration of putative TRDs of monospecific MTases, pointing to differences between multi- and monospecific MTases in the way their core and TRD sequences interact.

Base Sequence↗
PubMed1992

Chemo-adoptive immunotherapy of nude mice implanted with human colorectal carcinoma and melanoma cell lines.

The antitumor effects of chemotherapy, recombinant human interleukin-2 (IL-2), recombinant human interferon alpha A/D (IFN alpha), allogeneic human lymphokine-activated killer (LAK) cells, and antitumor monoclonal antibody (mAb), administered alone and in various combinations, were tested in athymic nude mice carrying human tumor xenografts. Treatment began 6-18 days after i.v. or i.p. inoculation of colorectal carcinoma or melanoma cell lines, when macroscopic growths were evident. Chemotherapy consisted of two or three courses of 5-fluorouracil (5-FU) or dacarbazine. IL-2 and/or IFN alpha were administered three to five times weekly for 1-3 weeks, usually starting 2-5 days after chemotherapy. Human LAK cells were infused once or twice weekly for 2 or 3 weeks concurrently with IL-2. In some experiments, murine anticolorectal carcinoma mAb (SF25) was administered. In both tumor systems, chemotherapy alone or immunotherapy alone (IL-2, IL-2 + LAK cells, IFN alpha, IL-2 + IFN alpha +/- LAK cells) had little or no therapeutic effects. Additive effects were obtained by combining chemotherapy with IL-2 and LAK cells or with IL-2 and IFN alpha. In the majority of the experiments, the most effective combination was chemotherapy + IL-2 + IFN alpha + LAK cells. Treatment with mAb was beneficial in the colorectal carcinoma system when combined with 5-FU + IL-2 or 5-FU + IL-2 + IFN alpha. Homing experiments with radiolabeled human and mouse LAK cells injected i.v. showed increased early accumulation in the liver and lungs, whereas freshly explanted mouse splenocytes localized mostly in the spleen and liver. The tissue distribution pattern of human LAK cells was similar in normal and tumor-bearing mice (with lung metastases). These findings suggest that combination of chemotherapy with cytokines and LAK cells can be partially effective for advanced solid human tumors even in the absence of the host's T-cell immune response. Preliminary experiments showed that tumor-specific, anti-melanoma T-cell clones were effective in local (s.c.) tumor growth inhibition (Winn assay) following coinjection with the autologous tumor cells.

Animals↗
PubMed1992

Experimental approaches to guarantee minimal risk of potential virus in purified monoclonal antibodies.

Regarding biological products, increasing awareness of potential side effects have placed great importance not only at protein purity regarding other proteins but on the removal of biologicals such as DNA and especially virus the importance of which may not be known. Monoclonal antibodies (Mab) have come to be an important class of molecules obtained from hybridoma cells, i.e., nonrecombinant cells in culture. It has been noted during the last years, that with rare exceptions hybridoma cell lines contain retrovirus like particles. The infectious nature of the EM-visible particles has been tested for, however, in most cases not been substantiated. In order to bring these valuable biological reagents, Mab's, to good use in man for imaging or therapy, the remaining concern about a potential retroviral infection has to be reduced to an acceptable minimum. We describe experimental approaches for the validation of chromatographic and ultrafiltration steps used in the production of monoclonal antibodies to remove and inactivate murine retrovirus. Present day biotechnological manufacturing processes have been devised incorporating a number of strategic preventive measures that have found wide spread acceptance. They permit to answer the question: how can a potentially harmful infection by an unknown virus be excluded. Knowledge of the efficacy of purification steps to clear infectious model virus is fundamental to devise biotechnological manufacturing processes yielding a purified antibody for use in man.

Animals↗
PubMed1992

The integration of computed tomography and magnetic resonance imaging in treatment planning for gynecologic cancer.

Both CT and MRI may play an important role in the initial assessment and subsequent management of patients with gynecologic cancer. The different capabilities of these examinations make the choice of test dependent on what portion of the body is visualized (e.g., chest, upper abdomen, or pelvis) and what information is sought. Body imaging can be scheduled appropriately when the findings from the study will have a significant impact on patient management (e.g., operative versus nonoperative management, selection of treating physician, and initial modality of treatment). However, the limitations of each modality must be appreciated. In general, a negative CT or MRI will not exclude the possibility of small volume disease involvement, and a critical positive study should, when practical, be histologically confirmed. Neither test stands alone, but each plays a role when integrated with careful clinical history, physical examination, and other laboratory and radiologic examinations.

Endometrial Neoplasms↗
PubMed1992

Evaluation of the vertebrobasilar-posterior system by transcranial color duplex sonography in adults.

BACKGROUND AND PURPOSE: The transcranial color duplex sonography technique was applied to the vertebrobasilar-posterior system to provide normal data for clinical application. METHODS: The intracranial posterior circulation was studied in 49 healthy volunteers (mean +/- SD age, 35 +/- 12 years) by a transcranial and suboccipital approach with a 2.0-MHz sector transducer of a computed sonography system. RESULTS: The posterior cerebral artery and the vertebrobasilar system were depicted clearly in the color Doppler mode. Pulsed Doppler signals could be recorded in the posterior cerebral (100%), basilar (92%), and vertebral arteries (89%). The following normal values were provided for all vessels: systolic peak, end-diastolic maximum, time-averaged, and time-averaged maximum velocities; resistance and pulsatility indexes; and a spectral broadening index. Mean +/- SD values were 45.9 +/- 9.6, 45.5 +/- 10.8, and 39.2 +/- 10.6 cm/sec for time-averaged maximum velocity, and 28.3 +/- 6.5, 30.6 +/- 7.2, and 24.7 +/- 8.4 cm/sec for time-averaged velocity in the posterior cerebral, basilar, and vertebral arteries, respectively. In a reproducibility study, duplex measurements of the posterior cerebral arteries were repeated in 27 subjects. The correlation between the two examiners was high (r = 0.56, p less than or equal to 0.0001 for time-averaged maximum velocity). CONCLUSIONS: Color duplex sonography of the vertebrobasilar-posterior system is a new, noninvasive, bedside investigative technique. It permits visualization of artery flow in real time, relating these to adjacent brain and cranial structures, as well as angle-corrected duplex measurement of "true" flow velocities at defined sites of the vessels. Thus, it will open new diagnostic possibilities in disorders of the posterior circulation.

Adult↗
PubMed1992

The effects of local anesthetics containing epinephrine on digital blood perfusion. 1978.

Digital perfusion research was conducted with two local anesthetics, lidocaine and bupivacaine. The studies were performed utilizing both anesthetic agents, plain and with various concentrations of epinephrine. The drugs' effects on blood perfusion over a 24-hr. period were recorded and discussed. The onset and duration of anesthesia were also compared.

Anesthesia, Local↗
PubMed1992

Radiographic evidence of disease progression in methotrexate treated and nonmethotrexate disease modifying antirheumatic drug treated rheumatoid arthritis patients: a meta-analysis.

Methotrexate (MTX) has proven to be efficacious in the treatment of rheumatoid (RA), but it remains to be proven whether it can slow disease progression, as determined radiographically, in comparison with other disease modifying antirheumatic drugs (DMARD). We performed a meta-analysis of the available data to answer this question. A literature search, including abstracts, was conducted and inclusion criteria developed (description of patients, accountability of patients, inclusion of a control group of patients, specified radiographic endpoint, and appropriate reading of the radiographs). Publications were scored on a scale of 0 to 5 with a score > or = 3 required for inclusion in the study. For abstracts selected, additional data were obtained directly from the investigators. Data for 353 MTX treated and 205 non-MTX-DMARD treated patients with RA were gathered. Not all publications used the same scoring system, so some assumptions were required to analyze the combined data. Only the erosion score was included since not all publications included a reading of the joint space. All scores were transformed into Sharp scores (Arthritis Rheum 1985;28:1449), including the important contributions of 3 Larsen scored publications. Finally a monthly rate of disease progression was computed. Several comparisons were made. Overall, the rates of disease progression were similar for MTX and non-MTX-DMARD treated patients with RA. The non-MTX-DMARD treated patients with RA were separated into a group treated with gold salts (oral or parenteral) and a group treated with azathioprine with each group compared to the MTX treated patients. MTX had slower rates of disease progression than azathioprine, (rates 0.004 vs 0.012) but not slower rates than gold salts (0.008 vs 0.008). Despite its efficacy, the possible role of MTX in slowing disease progression more than other DMARD, as determined radiographically, appears to be evident only when compared to azathioprine.

Adult↗
PubMed1991

Cloning, characterization and evolution of the BsuFI restriction endonuclease gene of Bacillus subtilis and purification of the enzyme.

The restriction endonuclease (R.BsuFI) of Bacillus subtilis recognizes the target DNA sequence 5' CCGG. The R.BsuFI gene was found in close proximity to the cognate M.BsuFI gene, which had previously been characterized (1). Cloning of the R.BsuFI gene in E.coli was only possible with the M.BsuFI Mtase gene present on a compatible plasmid. The cloned R.BsuFI gene was expressed in E. coli and restriction activity was observed in vivo and in vitro. The R.BsuFI gene consists of 1185 bp, coding for a protein of 395 amino acids with a calculated molecular weight of 45.6 kD. The R.BsuFI enzyme was purified to homogeneity following overexpression. It presumably works as a dimer and cleaves the 5' CCGG target sequence between the two cytosines to produce sticky ends with 5' CG overhangs, like the isoschizomers R.MspI and R.HpaII. The relatedness between R.BsuFI and R.MspI is reflected by significant similarities of the amino acid sequences of both enzymes. This is the first case where such similarities have been observed between isoschizomeric restriction endonucleases which belong to 5mC specific R/M systems. This observation suggests that R.BsuFI and R.MspI genes derive from a common ancestor. In spite of such functional and evolutionary relatedness, the R/M systems differ in the arrangement of their R and M genes. In the BsuFI system transcription of the two genes is convergent, whereas divergent transcription occurs in the MspI system.

Amino Acid Sequence↗
PubMed1991

Regenerating retinal axons of goldfish respond to a repellent guiding component on caudal tectal membranes of adult fish and embryonic chick.

On a substrate of rostral/caudal tectal membrane stripes of adult fish, regenerating temporal retinal axons avoid the caudal membranes. Thus they behave like embryonic chick axons on chick E9 membranes. The caudal membranes of adult fish contain a repellent component that, as has previously been shown in the chick, is inactivated by the enzyme PI-PLC. Fish axons respond not only to their own but also to the repellent component of embryonic chick membranes. Fish and more so chick E9 caudal membranes have an outgrowth reducing effect on fish axons that is also abolished by PI-PLC treatment and is weaker on chick E16 membranes. Thus adult fish tecta express a guiding component for retinal axons related to that in the embryonic chick.

Animals↗
PubMed1991

'Pseudo' domains in phage-encoded DNA methyltransferases.

5-Cytosine-DNA-methyltransferases, which are found in many organisms ranging from bacteriophages to mammals, transfer a methyl group from S-adenosylmethionine to the carbon-5 of a cytosine residue in specific DNA target sequences. Some phage-encoded methyltransferases methylate more than one sequence: these enzymes contain several independent target-recognizing domains each responsible for recognizing a different site. The amino-acid sequences of these multispecific methyltransferases reveal that some enzymes in addition carry domains that do not contribute to the enzymes' methylation potential, but strongly resemble previously identified target-recognizing domains. Here we show that introducing defined amino-acid alterations into these inactive domains endows these enzymes with additional methylation specificities. Gel retardation analysis demonstrates that these novel methylation specificities correlate with the acquisition of additional DNA-binding potential of the proteins.

Amino Acid Sequence↗
PubMed1991

Molecular mechanisms separating two axonal pathways during embryonic development of the avian optic tectum.

During embryonic development of the avian optic tectum, retinal and tectobulbar axons form an orthogonal array of nerve processes. Growing axons of both tracts are transiently very closely apposed to each other. Despite this spatial proximity, axons from the two pathways do not intermix, but instead restrict their growth to defined areas, thus forming two separate plexiform layers, the stratum opticum and the stratum album centrale. In this study we present experimental evidence indicating that the following three mechanisms might play a role in segregating both axonal populations: Retinal and tectobulbar axons differ in their ability to use the extracellular matrix protein laminin as a substrate for axonal elongation; the environment in the optic tectum is generally permissive for retinal axons, but is specifically nonpermissive for tectobulbar axons, resulting in a strong fasciculation of the latter; and growth cones of temporal retinal axons are reversibly inhibited in their motility by direct contact with the tectobulbar axon's membrane.

Animals↗
PubMed1991

[Effect of gastrointestinal hormones on duodenal motility in acute experiments].

In this study was observed the influence of Pancreozymin, Secretin, Glucagon, Pentagastrin and Somatostatin on the motility of duodenum in 256 seances in acute experiments on 12 dogs before and after truncal vagotomy and on 20 Wistar rats. As a control we used the return of the parameters of electrical and motor activity to their original levels. The reaction was registered with the help of electromyography, using bipolar electrodes and with measurement of intraluminal pressure, using open-tip catheter technique. Spike-activity was graphically presented and statistically performed. It was shown, that Secretin and Glucagon caused relaxation of duodenum, but Pentagastrin, Pancreozymin and Somatostatin stimulated duodenal motility with spastic component and gradual phasic character of a process. After truncular vagotomy the reaction to Pancreozymin and Pentagastrin was slowly, to Glucagon strongly reduced, but the reaction to Secretin was extended. We observed after TV either no reaction or a diminished reaction to Somatostatin. This study serves as experimental basis for use of gastrointestinal hormones and electromyography for diagnosis and therapy of motility disorders of duodenum in surgical practice.

Animals↗
PubMed1991

[Invasive, metastatic growth of lymphoblastoid B cells in immunodeficient SCID mice].

A Burkitt's lymphoma (BL, EBV +, 8/22 translocation), the EBV immortalized lymphoblastoid cell line from the same individual (LCL) and somatic cell hybrids (HYB) between both cell lines were inoculated in immunedeficient scid mice. Subcutaneous injected BL cells produced local tumor masses without distant metastases. In contrast LCL and hybrids show invasive and disseminated growth. Peripheral lymph nodes and thymic tissue were preferentially colonized. A detailed phenotypic analysis of BL, LCL and 3 different hybrids was performed using differntiation and adhesion molecules. The preferential "homing" of LCL and hybrids seem to be correlated with the expression of the lymphocyte homing receptor (CD44). The growth of LCL in scid mice shows, that additional genetic alterations are not necessary to cause a malignant phenotype. Activation of myc seem to play a minor role, because hybrids mimic the LCL phenotype and distribution pattern.

Animals↗