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PubMed1997

Behavioral comparison of the oximes TMB-4, 2-PAM, and HI-6 in rats using operant conditioning.

It has recently been shown that oximes can amplify the ability of cholinesterases to scavenge organophosphorus (OP) agents. Since both OP agents and oximes can disrupt performance, behavioral evaluation of bioscavenger therapies using oximes can be hindered. Therefore, we investigated the ability of three oximes, administered alone, to disrupt performance. The effects of trimedoxime bromide (TMB-4) (3.16-56.2 mg/kg), pralidoxime chloride (2-PAM) (10.0-237.1 mg/kg), and, 1-([[4-amincarbonyl)pyridino]-methoxy]-methyl)-2, 4-bis[(hydroxyimino)methyl] pyridinium dichloride monohydrate (HI-6) (10.0-237.1 mg/kg) were evaluated in rats using a variable-interval 56 (VI 56) s schedule of food reinforcement. Under control conditions, the VI 56 s schedule produced a constant rate of responding (i.e., lever-pressing). All three oximes produced dose-dependent decreases in responding, and the largest doses of TMB-4 and 2-PAM produced complete or nearly complete suppression of responding in all rats. Only the largest dose of HI-6 suppressed responding. Analysis of the dose-effect functions demonstrated that TMB-4 was substantially more potent than 2-PAM, which was slightly more potent than HI-6, for producing response suppression. These results establish doses of each oxime that will not contribute to disruption of responding, and thus, facilitate future evaluation of bioscavenger therapies against OP toxicity.

Animals↗
PubMed1999

Synthesis and characterization of complexes of p-isopropyl benzaldehyde and methyl 2-pyridyl ketone thiosemicarbazones with Zn(II) and Cd(II) metallic centers. Cytotoxic activity and induction of apoptosis in Pam-ras cells.

It is known that metallic complexes of methyl 2-pyridyl ketone thiosemicarbazone (HL1) and p-isopropyl benzaldehyde thiosemicarbazone (HL2) may have potential antitumor activity. We have prepared complexes of HL1 and HL2 with Zn(II) and Cd(II). The cytotoxic activity shown by these compounds against cell lines sensitive and resistant to cis-diamminedichloroplatinum(II) (cis-DDP) indicates that coupling of HL1 and HL2 to Zn(II) and Cd(II) centers may result in metallic complexes with important biological properties since they display IC50 values in a microM range similar to that of the antitumor drug cis-DDP. Moreover, it is interesting to note that the Zn/HL2 complex exhibits specific cytotoxic activity against Pam-ras cells (cis-DDP resistant cells which over-express the H-ras oncogene) with an in vitro therapeutic index of 3.26 versus 0.78 for cis-DDP. Treatment of Pam-ras cells with the IC50 value of the Zn/HL2 compound induces a 'DNA ladder' (fragmentation of genomic DNA in nucleosome units) indicative of apoptosis in this ras-transformed cell line. In contrast, a 'DNA smear' (non-specific fragmentation of genomic DNA) is observed in Pam 212 normal cells treated with the IC50 of this compound. The analysis by circular dichroism (CD) spectroscopy of the interaction of the Zn/HL2 compound with calf thymus DNA (CT DNA) indicates that it produces stronger alterations on the double helix conformation than cis-DDP. So, these results suggest that Zn/HL2 may be considered a potential antitumor agent.

Animals↗
PubMed2003

Solids, organic load and nutrient concentration reductions in swine waste slurry using a polyacrylamide (PAM)-aided solids flocculation treatment.

Increased swine production results in concentration of wastes generated within a limited geographical area, which may lead to land application rates exceeding the local or regional assimilatory capacity. This may result in pollutant transfer through surface water or soil-groundwater systems, environmental degradation, and/or odor concerns. Existing swine waste pit storage and lagoon treatment technologies may be inadequate to store or treat waste prior to land application without these concerns resulting. Efficient swine waste solids separation may reduce environmental health concerns and generate a value-added bioresource (solids). This study evaluated the efficiency of a polyacrylamide (PAM) flocculant-aided solids separation treatment to reduce pollution indicator concentrations in raw (untreated) swine waste slurry. Swine waste slurry solids separation efficiency through gravity settling (sedimentation) was evaluated before and after the addition of a proprietary polymeric (PAM) flocculant. Results indicated that polymer amendments at concentrations of 62.5-750 mg/l improved slurry solids separation efficiency and significantly reduced concentrations of other associated aquatic pollution indicators in a majority of analyses conducted (33 of 50 total analyses conducted). Results also suggested that PAM-aided solids separation from swine waste slurry might facilitate further treatment and/or disposal and therefore reduce associated environmental degradation potential.

Acrylic Resins↗
PubMed2006

Novel transplatinum(II) complexes with [N2O2] donor sets. Cellular pharmacology and apoptosis induction in Pam 212-ras cells.

Cellular pharmacological properties of eight trans-picoline platinum(II) complexes of formula trans-[PtX(2)(L)(L')], where X = Cl or CH(3)COO (OAc) and L = L' = 3-picoline (3-pic), 4-picoline (4-pic) or L = NH(3) and L' = 3-pic or 4-pic, were investigated in murine keratinocyte Pam 212 cells and Pam 212-ras cells, murine tumor keratinocytes derived from transformation with a viral vector containing the H-ras oncogene. The derivatives trans-[Pt(OAc)(2)(L)(L')] (L = L' = 3-pic, 9, and L = L' = 4-pic, 10) were able to circumvent resistance in Pam 212-ras cells. Although all the trans-picoline platinum(II) acetate derivatives showed a similar level of DNA binding, there were remarkable differences in cellular accumulation: the complexes having two picoline ligands (9, 10) had a much higher intracellular accumulation than those having mixed picoline and ammine ligands (11, 12). No significant differences in cellular pharmacological properties have been observed between isomers having 3- or 4-picoline.

Animals↗
PubMed2000

Substance P induction of murine keratinocyte PAM 212 interleukin 1 production is mediated by the neurokinin 2 receptor (NK-2R).

The neurological system plays an important role in modulating some inflammatory skin diseases. Neuro-cutaneous interactions may be mediated by the release of neuropeptides such as substance P (SP) which activate immunocompetent cells in the skin by binding to high affinity neurokinin receptors (NKR). Since epidermal keratinocytes produce a variety of cytokines and are intimately associated with cutaneous sensory fibers, we tested the ability of these cells to participate in the cutaneous neuroimmune system by the secretion of potent cytokines such as interleukin 1 (IL-1) in response to released SP. RT-PCR studies demonstrated that cultured PAM 212 murine keratinocytes expressed mRNA for NK-2R but not NK-1R. Correspondingly, the addition of SP to these cells resulted in a rapid increase in intracellular Ca2+ levels that could be specifically blocked by an NK-2R antagonist. NK-2R was also shown in normal mouse epidermis by immunohistochemistry. SP augmented the expression of PAM 212 keratinocyte IL-1alpha mRNA in a dose and time dependent manner and this induction was inhibited by an NK-2R antagonist. Secretion of bioactive IL-1alpha by the PAM 212 keratinocytes was likewise stimulated by SP in a dose dependent manner. These data support the hypothesis that SP released from cutaneous sensory nerves contributes to neuroimmune inflammatory responses in the skin by modulating the expression and release of cytokines from epidermal keratinocytes.

Animals↗
PubMed1993

A fourth class of theta-replicating plasmids: the pAM beta 1 family from gram-positive bacteria.

Plasmid pAM beta 1 from Enterococcus faecalis uses a unidirectional theta mode of replication. We show here that this replication (i) is dependent on a plasmid-encoded replication protein (Rep) but not on a DNA structure typical for origins of most Rep-dependent plasmids and (ii) is initiated by DNA polymerase I (PolI). pAM beta 1 minimal replicon shares no homology with highly conserved ColE1-type replicons, which use PolI for initiation but do not encode a Rep, or with ColE2 and ColE3 replicons, which require PolI for replication and encode a Rep. We propose that pAM beta 1 and a number of other naturally occurring and closely related plasmids from a distinct plasmid class.

Base Sequence↗
PubMed1984

Acute malathion toxicosis and related enzymatic alterations in Bubalus bubalis: antidotal treatment with atropine, 2-PAM, and diazepam.

Oral administration of malathion (MTH) in sublethal (100 mg/kg) or minimal lethal (125 mg/kg) doses in buffalo calves produced toxicity with an onset within 15-20 min and peak effects including severe tremors and convulsions within 40-60 min. Various antidotal drugs were administered alone or in combination at the time of peak malathion toxicity (within 1 h) and were assessed for their ability to alleviate signs of cholinergic toxicity. Blood cholinesterase and aminotransferases activities were monitored at various times. A combination of atropine sulfate (0.5 mg/kg, 1/4 iv and 3/4 im) and pyridine 2-aldoxime methiodide (2-PAM, 20 mg/kg, iv) reversed the clinical evidence of malathion toxicity within 15 min. The combination of atropine sulfate and diazepam (0.75 mg/kg, iv) prevented death and cholinergic signs of toxicity except for weak muscular fasciculations, which persisted for 30-60 min. Atropine sulfate alone was less effective and also did not reverse malathion-induced biochemical changes. In contrast, administration of either 2-PAM (10-30 mg/kg, iv) or diazepam (0.5-1.0 mg/kg, iv) alone accentuated malathion toxicity. Thus, the combination of atropine sulfate and 2-PAM was the most effective antidotal treatment in acute malathion toxicity.

Animals↗
PubMed1985

On the PAM matrix model of protein evolution.

The internal consistency of the PAM matrix model of protein evolution is here investigated. The 1 PAM matrix has been constructed from amino acid replacements observed in closely related sequences. Such replacements are of two types, those that do not require an intermediate amino acid replacement and those that do. The second type of replacement must generally be produced by a repetition of the first. This allows data on the first type to be used in predicting data on the second type so that some elements of the 1 PAM matrix may be used to predict others. A discrepancy of more than two orders of magnitude is found between the predictions and the data when this is carried out. This is partly accounted for by an error in constructing the matrix. However, it also seems necessary that the basic model be modified. Several possibilities are considered. One of these is to incorporate a site-dependent spectrum of mutabilities associated with each amino acid.

Amino Acid Sequence↗
PubMed1993

Mouse dendritic epidermal T cells exhibit chemotactic migration toward PAM 212 keratinocyte culture supernatants.

Dendritic epidermal T cells (DETCs) are Thy-1+, CD45+, CD3+, CD4-, CD8-, and T-cell receptor-V gamma 3/V delta 1+ leukocytes that reside normally in adult mouse skin. We have demonstrated previously that keratinocytes serve as adhesion substrates for DETCs, and that interleukin 7 (IL-7), which is produced by keratinocytes, serves as a growth factor for DETCs. The present study was conducted to address the mechanisms by which DETCs migrate into the epidermis, reasoning that keratinocytes may also be a source of chemotactic activity. Short-term DETC lines were 35S-labeled and tested for migration toward Pam 212 keratinocyte culture supernatants using a modified Boyden chamber method; cell movement from upper chambers toward test samples in lower chambers was traced by counting radioactivity. DETC displayed rapid (within 60 min) and marked (> 50%) migration toward keratinocyte supernatants. The majority of cells that had migrated into keratinocyte supernatants expressed the V gamma 3 T-cell receptor, thus verifying that the migrating cells were DETCs. Addition of keratinocyte supernatants to the upper chambers completely blocked migration, suggesting its chemotactic nature. By contrast, no DETC migration was observed toward 3T3 fibroblast supernatants. Chemotactic activities were 1) produced by Pam 212 cells even in the absence of serum; 2) greater than 12 kD in size; 3) heat and pH labile; 4) trypsin sensitive; and 5) precipitated by 60-100% ammonium sulfate. Several cytokines (e.g., IL-1 alpha and IL-8) failed to mediate DETC migration when added to the lower chambers. Likewise, the same cytokines, when added to the upper chambers, failed to inhibit DETC migration toward Pam 212 supernatants. These results support our hypothesis that keratinocytes facilitate the residence of DETC in epidermis by secreting unique chemotactic factors, by providing adhesion substrates, and by elaborating specific growth factors.

Animals↗
PubMed1991

Regulation of GM-CSF and IL-3 production from the murine keratinocyte cell line PAM 212 following exposure to ultraviolet radiation.

Ultraviolet radiation (UVR) exposure induces profound changes in the synthesis and secretion of various cytokines both in vivo and in vitro. Little is known regarding the mechanism of these responses. This investigation evaluated the effects of UVR on the ability of a murine keratinocyte line (PAM 212) to produce interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF). Subconfluent rapidly dividing PAM 212 cells were shown by RNA slot-blot hybridization studies to have increased levels of mRNA for both IL-3 and GM-CSF within 1 h of UVR exposure. However, only GM-CSF-specific bioactivity, as determined by antibody neutralization studies, was shown to increase above baseline in cell supernatants. Cells grown to confluence responded differently to UVR. Under these culture conditions an apparent decrease in bioactivity was detected after UVR exposure for both growth factors, and no change in mRNA levels was detected. In addition to culture density, removal of extracellular calcium or sodium during irradiation, treatment with amiloride, or inhibition of new mRNA synthesis with cordycepin was shown to influence the UVR-induced alteration in release of IL-3 or GM-CSF bioactivity from both confluent and subconfluent PAM 212 cells. These results demonstrate that UVR influences the release of the colony stimulating factors GM-CSF and IL-3 from keratinocyte, and suggests that the state of cell growth and conditions of membrane ion transport influence the mechanisms regulating secretion of those factors.

Animals↗
PubMed1986

Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum.

The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.

Conjugation, Genetic↗
PubMed1996

Distribution of peptidyl-glycine alpha-amidating mono-oxygenase (PAM) enzymes in normal human lung and in lung epithelial tumors.

C-terminal alpha-amidation is a post-translational modification necessary for the biological activity of many regulatory peptides produced in the respiratory tract. This modification is a two-step process catalyzed by two separate enzyme activities, both derived from the peptidyl-glycine alpha-amidating mono-oxygenase (PAM) precursor. The distribution of these two enzymes, peptidyl-glycine alpha-hydroxylating monoxygenase (PHM) and peptidyl-alpha-hydroxyglycine a amidating lyase (PAL), was studied in the normal lung and in lung tumors using immunocytochemical methods and in situ hybridization. In normal lung the enzymes were located in some cells of the airway epithelium and glands, the endothelium of blood vessels, some chondrocytes of the bronchial cartilage, the alveolar macrophages, smooth muscle cells, neurons of the intrinsic ganglia, and in myelinated nerves. A total of 24 lung tumors of seven different histological types were studied. All cases contained PAM-immunoreactive cells with various patterns of distribution. All immunoreactive cells were positive for the PHM antiserum but only some of them for the PAL antiserum. The distribution of PAM co-localizes with some other previously described amidated peptides, suggesting that amidation is an important physiological process taking place in the normal and malignant human lung tissue.

Aged↗
PubMed2004

CFR-1 receptor as target for tumor-specific apoptosis induced by the natural human monoclonal antibody PAM-1.

The human monoclonal antibody PAM-1 was isolated from a patient with stomach cancer. The germ-line coded IgM antibody identifies a recently described 130 kDa variant of CFR-1 (cysteine-rich fibroblast growth factor receptor 1). This CFR-1/PAM-1 receptor is post-transcriptionally modified and over-expressed on human epithelial tumors and carcinoma pre-cancer lesions such as H. pylori induced gastritis, intestinal metaplasia and dysplasia of the stomach, ulcerative colitis-related dysplasia and adenomas of the colon, Barrett metaplasia and dysplasia of the esophagus, squamous cell metaplasia and dysplasia of the lung and cervical intraepithelial neoplasia. Furthermore, the expression of CFR-1/PAM-1 correlates with the proliferation rate and increases with the grade of malignancy. This study demonstrates that the human monoclonal antibody PAM-1 inhibits cell growth and induces apoptosis, in vitro and in vivo. Both, the unique tumor-specific expression of the CFR-1/PAM-1 receptor and the growth inhibitory effect of the PAM-1 antibody makes this combination a good diagnostic and therapeutic tool for all kinds of epithelial cancers and precursor lesions.

Adenocarcinoma↗
PubMed2004

PAM-1, a natural human IgM antibody as new tool for detection of breast and prostate precursors.

Early detection and differential analysis of premalignant lesions are very important for both prognosis and therapy of cancer patients. A good source of diagnostic tools is the natural antibody pool of humans. Tumor-specific antibodies can be established by using hybridoma technology. The fully human germline-coded monoclonal IgM antibody PAM-1 was isolated from a patient with a stomach carcinoma. PAM-1 reacts with a post-transcriptionally modified isoform of membrane receptor CFR-1 which is overexpressed on almost all epithelial cancers of all types and origins. The expression of CFR-1/PAM-1 on precancerous stages of breast and prostate cancer was analyzed by immunohistochemistry and compared with normal breast and prostate tissue as well as adenocarcinomas of both. In addition FACS analysis was performed to detect receptor expression on benign and malign prostate cells. 73 different tissue samples of prostate and breast precancerous stages and prostate and breast carcinomas were analysed for CFR-1/PAM-1 expression immunohistochemically. The CFR-1/PAM-1 receptor was expressed on nearly all precancerous stages and carcinomas while normal breast and prostate tissue showed negative results. These results were confirmed by FACS analysis showing a CFR-1/PAM-1 expression only on prostate carcinoma cells but not on benign prostate hyperplasia cells. The unique expression of this new CFR-1/PAM-1 receptor makes the PAM-1 antibody an ideal diagnostic and even therapeutic tool for precancerous and cancerous epithelial lesions of the breast and the prostate.

Antibodies↗
PubMed2004

[The ht-PAm cDNA knock-in the goat beta-casein gene locus].

The production of recombinant protein is one of the major successes of biotechnology, animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animal mammary gland bioreactor are being used for this purpose. Gene targeting is a more powerful method to produce mammary gland bioreactor, and nuclear transfer from cultured somatic cells provides an wonderful means of cell-mediated transgensis. Here we describe efficient and reproducible gene targeting in goat fetal fibroblasts to place the human tissue plasminogen activator mutant (ht-PAm) cDNA at the beta-casein locus, and would produce the transgenic goat by nuclear transfer. To construct the gene targeting vector pGBC4tPA, the milk goat beta-casein genomic DNA sequence for homologous arms had been cloned firstly. The left arm was 6.3 kb fragment including goat beta-casein gene 5' flanking sequence, and the right arm was 2.4 kb fragement including beta-casein gene from exon 8 to exon 9. The ht-PAm cDNA was subcloned in the goat beta-casein gene exon 2, and the endogenous start condon was replaced by that of ht-PAm. The bacterial neomycin (neo) gene as positive selection marker gene, was placed in the beta-casein gene intron 7, the thymidine kinase (tk) as the negative selection marker gene, was just outside the right arm. The validity of the positive-negative selection vector (PNS), was tested, and targeting homologous recombination (HR) were elevated to 5-fold with the negative selection marker using the drug GANC. The DNA fragment in which two LoxP sequence was delected effectively using Cre recombinase in vitro. Goat fetal fibroblasts were thawed and cultured to subconfluence before transfection, about 10(7) fibroblasts were electoporated at 240V, 600 microF in 0.8 mL PBS buffer containing linear pGBC4tPA. transfected cells were cultured in collagen-coated 96-wellplate for 24h without selection, then added the drug G418 (600 microg/mL) and GANC (2 micromol/L). After 12 days of selection, well separated G418r/GANCr clones were isolated and expanded in 24-wellplate. 244 clones were selected, and only 90 clones could grow and be tested by PCR screening for targeting. The primary result demonstrated that 31 targeting cell clones with homologous recombination events were obtained, and 2 cell clones was verified by DNA sequence analysis on the homologous recombination region.

Animals↗
PubMed1986

[Combination chemotherapy with cis-platinum, adriamycin and mitomycin C (PAM) in the treatment of non-small cell carcinoma of the lung].

Twenty-three patients with inoperable non-small cell lung cancer were treated with a combination chemotherapy of CDDP 100 mg/m2, ADM 30 mg/m2 and MMC 8 mg/m2 (PAM). Ten cases were adenocarcinoma, 9 cases were squamous cell carcinoma and 4 cases were large cell carcinoma. In 21 evaluable cases, partial response was obtained in 47.6%. (The response rates were 40.0% in patients with adenocarcinoma, 50.0% in those with squamous cell carcinoma and 66.7% in those with large cell carcinoma.) Leukocytopenia of less than 4,000/mm3 occurred in 100% of cases, thrombocytopenia of less than 100,000/mm3 occurred in 81.0%, and anemia(fall in hemoglobin over 2.0 g/dl) occurred in 66.7%. A transient elevation of Cr (over 1.5 mg/dl) and/or BUN (over 30 mg/dl) was observed in 23.8%. Nausea and vomiting occurred in almost all patients. No death occurred due to toxicity of PAM. These results demonstrate that PAM is an effective combination chemotherapy in patients with non-small cell carcinoma of the lung.

Adenocarcinoma↗
PubMed1987

Effects of atropine and 2-PAM chloride on vision and performance in humans.

These experiments assessed the time course and severity of effects of atropine and 2-PAM chloride on selected visual functions, physiological measures, and a tracking performance task. Atropine up to 4 mg X 70 kg-1 body weight and 2-PAM Cl in doses up to 1200 mg X 70 kg-1 body weight were administered intramuscularly in a double-blind Latin square design. A long lasting and dose-related increase in pupillary diameter and decrease in accommodative amplitude with accompanying loss of near visual acuity was found following atropine administration. These functions returned to baseline 2 d after injection. Distance acuity, contrast sensitivity, color vision, and intraocular pressure showed no changes after atropine. Tracking performance was significantly decreased by 4 mg of atropine. The changes in vision function cannot readily explain the tracking performance loss, since the tracking involved a distant visual target while the atropine produced degradation of near vision. Most of the physiological tests, tracking performance and all visual functions were unaffected by 2-PAM Cl.

Accommodation, Ocular↗