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Isolation of transformation-deficient Streptococcus pneumoniae mutants defective in control of competence, using insertion-duplication mutagenesis with the erythromycin resistance determinant of pAM beta 1.

Several transformation-deficient mutants of Streptococcus pneumoniae were isolated after insertion-duplication mutagenesis. Mutagenesis was accomplished by transformation of competent cells with chimeric DNA formed by the ligation of TaqI fragments of pneumococcal DNA to the erythromycin resistance determinant of the streptococcal plasmid pAM beta 1. The two mutants described were characterized as defective in the control of competence induction, possibly due to a block in the production of the intercellular competence-inducing protein.

Chimera↗

Factors affecting transfer frequency of pAM beta 1 from Streptococcus faecalis to Lactobacillus plantarum.

Conjugal transfer of streptococcal plasmid pAM beta 1 to Lactobacillus plantarum by a filter mating method was examined under various conditions. The transfer frequency depended greatly on the type, pore size, and side (front or back) of the membrane filter. The passage of sterilized water through the membrane under reduced pressure after donor and recipient cells were trapped on it increased the transfer frequency about 10-fold.

Conjugation, Genetic↗

A phase II study of combined adriamycin, L-PAM, and methotrexate with citrovorum factor rescue in advanced ovarian carcinomas.

In a phase II study of combined adriamycin, L-PAM, and methotrexate with citrovorum factor rescue, 14 of 15 patients with primary advanced or recurrent ovarian malignant tumors obtained an objective remission. In remission, all patients improved their quality of life. So far the median duration of remission is 5+ months for the complete responders and 7 months for the partial responders. Myelosuppression of varying severity occurred in 93% of the courses. The regimen proved highly effective, although toxic in some advanced patients.

Adult↗

Localization of amidating enzymes (PAM) in rat gastrointestinal tract.

We studied the distribution of the two enzymes involved in post-translational C-terminal alpha-amidation of regulatory peptides in rat digestive tract, using immunocytochemical methods and in situ hybridization techniques. The enzymes were located in most of the fibers and neurons of the myenteric and submucous plexus throughout the entire digestive tract and in endocrine cells of the stomach and colon. Staining of reverse-face serial sections demonstrated that the enzymes in endocrine cells of the stomach co-localized with gastrin in the bottom of the gastric glands. Some gastrin-immunoreactive cells near the neck of the gland were negative for PAM, suggesting that amidation takes place only in the more mature cells. In the colon all cells immunoreactive for glucagon and GLP1 were also positive for peptidylglycine alpha-hydroxylating monooxygenase (PHM) but not for peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The absence of immunoreactivity for the amidating enzymes in endocrine cells of the small intestine, known to produce C-terminally amidated peptides, suggests the existence of other amidating enzymes.

Animals↗

Immunocytochemical localization of peptidylglycine alpha-amidating monooxygenase enzymes (PAM) in human endocrine pancreas.

We studied the distribution of the enzymes that are involved in the post-translational alpha-amidation of regulatory peptides in human endocrine pancreas, using immunocytochemical methods for light and electron microscopy. Immunoreactivity for the two enzymes involved, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), was located in the periphery of the islets of Langerhans and in ductal endocrine cells. Staining of reverse-face serial sections demonstrated that these immunoreactivities co-localize with glucagon but not with pancreatic polypeptide (PP), insulin, or somatostatin. Double immunogold staining for electron microscopy confirmed the previous results and revealed a different localization for each enzyme inside the secretory granule: PHM is present in the central core of the glucagon-containing granules, whereas PAL is predominantly located near the granule membrane. The existence of an amidated peptide, GLP1, in the A-cells explains the presence of peptidylglycine alpha-amidating monooxygenase enzymes (PAM) in these cells. The absence of the enzymes in the PR-cells raises the possibility that a different form of amidating enzyme may be involved in the post-translational processing of this peptide.

Amidine-Lyases↗

Formation of the retinotectal projection requires Esrom, an ortholog of PAM (protein associated with Myc).

Visual system development is dependent on correct interpretation of cues that direct growth cone migration and axon branching. Mutations in the zebrafish esrom gene disrupt bundling and targeting of retinal axons, and also cause ectopic arborization. By positional cloning, we establish that esrom encodes a very large protein orthologous to PAM (protein associated with Myc)/Highwire/RPM-1. Unlike motoneurons in Drosophila highwire mutants, retinal axons in esrom mutants do not arborize excessively, indicating that Esrom has different functions in the vertebrate visual system. We show here that Esrom has E3 ligase activity and modulates the amount of phosphorylated Tuberin, a tumor suppressor, in growth cones. These data identify a mediator of signal transduction in retinal growth cones, which is required for topographic map formation.

Adaptor Proteins, Signal Transducing↗

Comparison of the photodynamic effect of exogenous photoprotoporphyrin and protoporphyrin IX on PAM 212 murine keratinocytes.

A comparative study of the cellular photosensitizing properties of protoporphyrin IX (PpIX) and photoprotoporphyrin (Ppp) was carried out in the transformed murine keratinocyte cell line, PAM 212. Time-course fluorescence studies were performed to determine the rate of uptake by cells together with fluorescence microscopy. The sensitized cells were laser irradiated with a range of light doses at 635 or 670 nm to determine the phototoxicity of the two compounds and to investigate their relative fluorescence photobleaching properties. Ppp showed enhanced phototoxicity at both its optimal activation wavelength of 670 nm (eight times more phototoxic than PpIX activated at its optimal wavelength of 635 nm for the same fluence) and at 635 nm (three times more phototoxic than PpIX at the same wavelength), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. The photobleaching rate of Ppp in cells was found to be higher using 670 nm irradiation compared with that of PpIX at 635 nm irradiation. At 635 nm, however, the photobleaching rate of Ppp was comparable to that of PpIX. The photobleaching quantum yields of the two compounds in cells were found to be similar at approximately 5 x 10(-4), with the same value confirmed at both 670 and 635 nm irradiation for Ppp. The fluorescence lifetime of Ppp in cells was measured as 5.4 ns using time-correlated single photon counting.

Animals↗

Evidence for intracellular aggregation of hypericin and the impact on its photocytotoxicity in PAM 212 murine keratinocytes.

We have assessed photoinduced toxicity of hypericin in PAM 212 murine keratinocytes and the relationship between concentration, incubation time and light fluence to evaluate the effect of intracellular aggregation at high concentrations. Confocal microscopy was used to establish the subcellular localization of hypericin at 5 and 50 microM and incubation times of 1 and 3 h. From fluorescence uptake time course studies, intracellular hypericin was demonstrated to exist predominantly in the monomeric form for up to 26 h incubation at 5 microM. However, there was a pronounced aggregation effect at 50 microM, with intracellular hypericin fluorescence levels initially showing an increase followed by a decrease with incubation time. This effect was subsequently shown to exert an effect on the phototoxicity of hypericin. On irradiation, the photocytotoxicity for 1 and 7 h incubation with 50 microM hypericin was comparable, whereas using 5 microM the photocytotoxicity showed good correlation with the intracellular fluorescence measurements at 1 and 7 h incubation.

Animals↗

Comparison by PAM fluorometry of photosynthetic activity of nine marine phytoplankton grown under identical conditions.

The photosynthetic activity of marine phytoplankton from five algal classes (Phaeodactylum tricornutum, Skeletonema costatum, Thalassiosira oceanica, Thalassiosira weissflogii, Dunaliella tertiolecta, Mantoniella squamata, Emiliania huxleyi, Pavlova lutheri and Heterosigma akashiwo) was investigated under identical growth conditions to determine interspecies differences. Primary photochemistry and electron transport capacity of individual species were examined by pulse amplitude-modulated (PAM) fluorescence. Although few differences were found in maximal photosystem II (PSII) photochemical efficiency between various species, large differences were noticed in their PSII-photosystem I (PSI) electron transport activity. We found that species such as T. oceanica and M. squamata have much lower photochemical activity than H. akashiwo. It appeared that processes involved in electron transport activity were more susceptible to change during algal evolution compared with the primary photochemical act close to PSII. Large variations in the nonphotochemical energy dissipation event among species were also observed. Light energy required to saturate photosynthesis was very different between species. We have shown that M. squamata and H. akashiwo required higher light energy (>1300 micromol m(-2) s(-1)) to saturate photosynthesis compared with S. costatum and E. huxleyi (ca 280 micromol m(-2) s(-1)). These differences were interpreted to be the result of variations in the size of light-harvesting complexes associated with PSII. These disparities in photosynthetic activity might modulate algal community structure in the natural environment where light energy is highly variable. Our results suggest that for an accurate evaluation of primary productivity from fluorescence measurements, it is essential to know the species composition of the algal community and the individual photosynthetic capacity related to the major phytoplankton species present in the natural phytoplankton assemblage.

Electron Transport↗

Multi-professional staff development programme for PAMs.

A unique, accredited, multi-professional education programme for junior staff in professions allied to medicine (PAMs) developed by Durham and Teesside Education and Training Consortium in collaboration with local trusts and social services is described. Designed to ensure competence to practice in line with clinical governance, it aims to strengthen multi-professional working practice through a greater understanding of roles and of the way the national health service (NHS) works. It enables staff to analyse their own learning needs and to employ self-directed learning skills for life-long learning. Using problem-based learning, it uses real clinical cases as triggers to promote critical evaluation and reasoning and the development of multi-professional care pathways.

Allied Health Personnel↗

Treatment of acute organophosphate poisoning: evidence of a direct effect on central nervous system by 2-PAM (pyridine-2-aldoxime methyl chloride).

Management of acute organophosphate poisoning in man includes rapid treatment with atropine and oximes. Oximes are thought to be unable to enter the central nervous system. We describe a case of parathion poisoning in a 3-1/2 year-old child and the effect of treatment with oxime 2-PAM (34 mg/kg) on EEG activity and clinical symptoms. The prompt improvement of cortical electrical activity documented by EEG could not be explained by any improvement of circulatory or respiratory function and has to be considered a direct effect of oximes on the central nervous system.

Brain↗

Efficient Secretion of Proteins Expressed from Insect Cells Directed by PAM Signal Peptide.

Signal and leading peptide sequences of rat PAM was inserted into the baculovirus transfer vector, and secretion expression plasmids pBACPAG2 and pBacPAI for the fusion gene PABC-hGRF and PABC-IGF-I were constructed, respectively. By cotransfection with linear genomic DNA of modified Autographa californica nuclear polyhedrosis virus (BacPAK6) and homologous recombination, the recombinant AcNPV, BacPAG and BacPAI, were obtained and identified. Fusion proteins PABC-hGRF and PABC-IGF-I were secreted efficiently from Sf21 cells infected with BacPAG and BacPAI, respectively, and those fusion protein could be purified efficiently by IgG affinity column.

Journal Article↗

High-pressure injection injury involving a 2-PAM chloride autoinjector.

High-pressure injection injuries to the hand are often associated with severe morbidity and should be considered surgical emergencies. The severity of these injuries is usually due to vascular compromise and the inflammatory nature of the material injected, such as paint, grease, and solvents. We present a case of a high-pressure injection of 2-PAM chloride into the finger of a 3-year-old boy. In this case, operative treatment was not necessary and all symptoms resolved.

Accidents, Home↗

[The PAM program for the early detection of breast cancer: the initial results (1987-1990). Programa de Detección Precoz de Alteraciones Mamarias].

We report the results of the PAM program for the early detection of breast cancer, carried out in Barcelona in females aged 50-70 years who were invited to participate by two personal letters. Out of 5435 invited females in the three years of the program, 37.2% responded to the first cycle and 70.8% to the second. The rate of detection of cancer was 6.9/1000 participating women in the first cycle. The validity of mammography was high, comparable to that from other countries. Sensitivity was 87.5%, specificity 99.1%, positive predictive value 45.2% and negative predictive value 99.9%. Letter would be insufficient as the only means of invitation to achieve a high participation rate in the first circle.

Age Factors↗

Physical characterisation of the replication region of the Streptococcus faecalis plasmid pAM beta 1.

The complete nucleotide (nt) sequence of a 5.1-kb EcoRI DNA restriction fragment carrying the replication region of the Streptococcus faecalis plasmid pAM beta 1 has been determined. Of the seven major open reading frames (ORF A-G) identified within this fragment, two (C and E) were shown to be encoding by in vitro transcription/translation assays. Evidence was obtained that synthesis of the polypeptide (Mr 57,380) encoded by the largest ORF (E) was essential for replication. Deletion analysis indicated that the minimum unit of DNA required for replication resided on a 2.59-kb AccI-HpaI subfragment. ORF C resided outside of this fragment and encompassed an extensive region of directly repeated nt sequence. The encoded polypeptide (Mr 30,471) was therefore composed of large tracts of reiterated amino acid sequence (11 x VDP and 35 x TEP tripeptides) which probably caused the observed anomalous electrophoretic mobility of the synthesised protein (equivalent to 61 kDa). Deletion of a 416-bp segment of DNA between unique KpnI and StyI sites caused an increase in copy number, which correlated with the in vitro production of higher levels of ORF E polypeptide. Although homology was detected between the sequenced DNA, and the replicon of a closely related streptococcal plasmid (pSM19035), none was evident to any other characterised Gram+ plasmid.

Amino Acid Sequence↗

[Analysis of prenatal care in adolescents who delivered in the obstetric clinic IPG PAM in Szczecin in the years 1990-1992].

The analysis of prenatal care was taken of 67 adolescent's who delivered in Obstetric Clinic IPG PAM in Szczecin, during the period 1990-1992. The quantitative dimension of prenatal care was evaluated by means of Kessner's Index at all, adapted by Buschner at all. The week of gestation that prenatal care began and number of prenatal visits were analysed. It was stated that adequate prenatal care received only 6% pregnant adolescents, inadequate 13.4%, intermediate 64% while 16.4% adolescents received no prenatal care. Earliest delivery and lowest infant birth weight were associated with no prenatal care. The significant correlation relationships between the week of the first visit, numbers of prenatal visits neonatal birth weight were no existed.

Adolescent↗