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Biomedical subjects

C A Thomas

Publications and source records attributed to C A Thomas.

At least 19 recordsLinked to original sources

Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils.

A pathological hallmark of Alzheimer's disease is the senile plaque, containing beta-amyloid fibrils, microglia and astrocytes. Beta-amyloid fibrils exert a cytotoxic effect on neurons, and stimulate microglia to produce neurotoxins, such as reactive oxygen species. Mononuclear phagocytes, including microglia, express scavenger receptors that mediate endocytosis of oxidized low-density lipoproteins, and adhesion to glucose-modified extra-cellular matrix proteins. Here we report that class A scavenger receptors mediate adhesion of rodent microglia and human monocytes to beta-amyloid fibril-coated surfaces leading to secretion of reactive oxygen species and cell immobilization. Thus, class A scavenger receptors are potential therapeutic targets in Alzheimer's disease.

Alzheimer Disease

Effects of two antiprogestins on early pregnancy in the long-tailed macaque (Macaca fascicularis).

The abortifacient effects of mifepristone and HRP 2000 were compared in gravid long-tailed macaques. Thirty-six animals were studied with treatment administered either by the oral (0.5 or 5.0 mg/kg; N = 5 per antiprogestin per dose) or intramuscular (i.m.) routes (0.5 mg/kg; N = 5 per antiprogestin) on gestational days (GD) 23-26; six vehicle controls were included. Blood samples were collected for assay of progesterone (P4) and each of the antiprogestins (pre-treatment, daily GD 23-28, every other day GD 30-40), and animals were monitored sonographically throughout gestation. Results of these studies indicated high rates of abortion with i.m. administration (3/5 mifepristone, 4/5 HRP 2000) and 5.0 mg/kg oral route (4/5, 2/5, respectively), with less effects noted at oral doses of 0.5 mg/kg (2/5, 0/5, respectively). No early abortions were observed in the control groups. Following daily i.m. treatment, peak levels of 8-16 ng/ml mifepristone were detected whereas 6-10 ng/ ml of HRP 2000 were noted (GD 26-27). No serum levels of mifepristone were detected following either of the oral doses whereas serum levels of 2-6 ng/ml HRP 2000 were noted with high dose oral administratation. Results of these studies suggest: (1) both antiprogestins are roughly comparable in terminating early pregnancy although HRP 2000 may be more efficacious when administered i.m. whereas mifepristone may be more effective when administered orally; (2) similar levels of biological activity are seen with the i.m. and high dose oral dosing regimens, with little or no activity with the oral low dose; and (3) infants resulting from surviving pregnancies were not affected by early gestation exposure.

Abortifacient Agents, Steroidal

Interplay between mycoplasmas and host target cells.

The infectious pattern of mycoplasmas (Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma genitalium) in mammalian cells was examined using confocal microscopy and flow cytometry combined with cell fractionation and mycoplasma viability determinations. Within 2 h postinfection mycoplasmas parasitize cell surfaces, enter the intracellular spaces and locate throughout the cytoplasmic and perinuclear regions. These mycoplasmas can be cultivated from cytoplasmic and nuclear fractions 96 h later and continue to persist intracellularly for at least 7 days, suggesting a much more active intracellular role for mycoplasmas than had been considered previously.

Animals

Role of the eosinophil in chronic vascular rejection of renal allografts.

Obiliterative arteriopathy in chronic renal allograft rejection is caused by intimal smooth muscle proliferation accompanied by infiltration of lymphocytes, monocytes, and eosinophils. We investigated the role of the eosinophil in chronic rejection. Twenty-four allograft nephrectomies were examined for the presence of eosinophils on hematoxylin-eosin-stained sections and using epifluorescence on Fisher-Giemsa-stained sections. Among 15 cases with chronic rejection, eosinophils were detected in 14 cases (93%) with epifluorescence compared with only six cases (40%) with hematoxylin-eosin staining (P = 0.005). With epifluorescence, eosinophils were identified in the intimal, adventitial, and tubulointerstitial compartments in 73%, 80%, and 87% of cases, respectively. To examine the pathogenic relevance of the eosinophils in the vessel wall, we investigated the effect of eosinophil-conditioned medium on DNA synthesis in cultured vascular smooth muscle cells. Autofluorescent eosinophils were isolated from atopic human donors using a fluorescence-activated cell sorter. Supernatant was collected from eosinophils (1 x 10(6)/mL) cultured overnight in medium with 0.5% fetal bovine serum. Incorporation of 3H-thymidine into DNA was measured in rat and human vascular smooth muscle cells treated for 24 hours with eosinophil-conditioned medium at 1:20, 1:10, 1:5, and 1:2 dilutions. Eosinophil-conditioned medium had a significant dose-dependent stimulatory effect on DNA synthesis in both cell lines. Our results indicate that eosinophil involvement in chronic renal allograft rejection is more common than previously recognized. The stimulatory effect of eosinophil-conditioned medium on vascular smooth muscle cell DNA synthesis suggests that eosinophils may be involved in the pathogenesis of the obliterative arteriopathy characteristically seen in chronic vascular rejection of renal allografts.

Animals

Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria offers certain advantages over other bioluminescence systems because no exogenously added substrate or co-factors are necessary, and fluorescence can be elicited by irradiation with blue light without exposing the cells producing GFP to invasive treatments. A mycobacterial shuttle-plasmid vector carrying gfp cDNA was constructed and used to generate transcriptional fusions with promoters of interest and to examine their expression in Mycobacterium smegmatis and Mycobacterium bovis BCG grown in macrophages or on laboratory media. The promoters studied were: (i) ahpC from Mycoosis and Mycobacterium leprae, a gene encoding alkyl hydroperoxide reductase which, along with the divergently transcribed regulator oxyR, are homologues of corresponding stress-response systems in enteric bacteria and play a role in isoniazid sensitivity; (ii) mtrA, an M. tuberculosis response regulator belonging to the superfamily of bacterial two-component signal-transduction systems; (iii) hsp60, a previously characterized heat-shock gene from M. bovis; and (iv) tbprc3, a newly isolated promoter from M. tuberculosis. Expression of these promoters in mycobacteria was analysed using epifluorescence microscopy, laser scanning confocal microscopy, fluorescence spectroscopy, and flow cytometry. These approaches permitted assessment of fluorescence prior to and after macrophage infection, and analyses of promoter expression in individual mycobacteria and its distribution within populations of bacterial cells. Bacteria expressing GFP from a strong promoter could be separated by fluorescence-activated cell sorting from cells harbouring the vector used to construct the fusion. In addition, the stable expression of mtrA-gfp fusion in M. bovis BCG facilitated localization and isolation of phagocytic vesicles containing mycobacteria. The experiments presented here suggest that GFP will be a useful tool for analysis of mycobacterial gene expression and a convenient cell biology marker to study mycobacterial interactions with macrophages.

Animals

Difficulties encountered in identification of a nutritionally deficient streptococcus on the basis of its failure to revert to streptococcal morphology.

Difficulties were encountered in the recognition of a nutritionally deficient streptococcus which continued to display aberrant morphologic forms (especially bulbous swellings and filament formation) despite provision of growth factors. With isolates displaying diverse morphologic entities not characteristic of a given species, e.g., Streptobacillus moniliformis or Erysipelothrix rhusiopathiae, nutritionally deficient streptococci should be considered.

Bacteremia

TAT-mediated transcellular activation of HIV-1 long terminal repeat directed gene expression by HIV-1-infected peripheral blood mononuclear cells.

We have previously shown evidence of Tat-mediated transcellular activation of the HIV-1 long terminal repeat (LTR) in vitro by using a laboratory-adapted strain of HIV-1. To examine the biologic significance of this observation, we asked whether primary PBMCs from HIV-1-infected individuals will transactivate the HIV-1 LTR transcellularly in suitable indicator cells. In cultures of PBMCs isolated from HIV-1-infected patients at various clinical stages, with either HIV-1 LTR-transfected Jurkat T cells or nonfusigenic HIV-1 LTR-transfected murine fibroblasts, transcellular activation was readily detected. Transactivation of the LTR in cocultured cells with HIV-1-infected PBMCs is detectable 1 to 2 wk before the onset of significant virus production and at ratios as low as 1 infected cell to 10(6) surrounding cells. Addition of the Tat inhibitor RO5-3335 substantially decreases transcellular activation, even at low concentrations (0.01 microM) that do not affect virus levels. In contrast, addition of the antiretroviral agent zidovudine has no effect on transcellular activation. These data suggest that Tat-mediated transcellular activation of the HIV-1 LTR occurs independently of cellular infection, and provides a mechanism that can promote the spread of HIV-1 in susceptible cell populations.

Cells, Cultured

Macrophages adhere to glucose-modified basement membrane collagen IV via their scavenger receptors.

Scavenger receptors have been reported to mediate macrophage adhesion to serum-coated plastic surfaces. We report here that scavenger receptors promote the divalent cation independent adhesion of human monocytes and macrophages to surfaces coated with non-enzymatically glycated collagen IV but not to surfaces coated with native collagen IV. Ligands for scavenger receptor types I and II blocked adhesion of monocytes and macrophages to non-enzymatically glycated collagen IV but had no effect on adhesion of these cells to albumin-coated surfaces. U937 human promonocyte-like cells transfected with cDNA encoding bovine scavenger receptor I or II adhered to surfaces coated with glycated-collagen IV but not to surfaces coated with native collagen IV. A synthetic peptide homologous to the domain of bovine scavenger receptor that binds modified low density lipoproteins (residues 327-343) inhibited the adhesion of U937 cells transfected with cDNA encoding bovine scavenger receptor II to glycated collagen IV, whereas a control peptide from the alpha helical domain of scavenger receptor II (residues 121-137) had no effect on adhesion of these cells. Macrophages plated on surfaces coated with glycated collagen IV were unable to endocytose acetylated low density lipoproteins from the medium, suggesting that their scavenger receptors were occupied in binding these cells to the substrate. These findings suggest new roles for scavenger receptors in the accelerated development of vascular lesions observed in diabetics.

Amino Acid Sequence

Clinical application of polymerase chain reaction amplification to diagnosis of herpes virus infection.

Amplification of viral DNA offers a potentially sensitive and specific method for identifying herpes viruses in pathologic specimens. The purpose of this study is to assess the value of polymerase chain reaction amplification of DNA as a diagnostic test for herpes virus in pathologic specimens. The purpose of this study is to assess for herpes virus infections. We examined 79 paraffin-embedded tissue samples from 43 patients and 55 viral culture samples from 45 patients. Herpes virus DNA in the specimens was amplified by polymerase chain reaction. Using paraffin-embedded tissue on which a diagnosis of herpes virus was made by morphologic criteria, 11 of 19 patients had herpes virus DNA identified by PCR; herpes virus DNA was not identified in any of 35 negative control specimens. Herpes virus DNA was also identified by polymerase chain reaction in all of the positive herpes virus culture specimens. Of 29 culture negative specimens, herpes virus DNA was identified in six. We conclude that polymerase chain reaction is useful to establish or confirm the presence of a herpes virus infection in paraffin-embedded tissue samples and that it is more sensitive than viral culture.

DNA, Viral

Host responses in patients with generalized refractory periodontitis.

Although patients with refractory periodontitis have been widely reported, no clear biologic profile of these patients has been noted. The purpose of the present study was to evaluate host responsiveness of a well-defined group of refractory periodontitis patients by determining the effect of a lipopolysaccharide (LPS) challenge on monocyte surface receptor density and on the release of inflammatory mediators. Venous blood was obtained from 7 refractory periodontitis, 8 stable periodontal maintenance, and 8 gingivitis patients with no evidence of periodontitis. Mononuclear cells were cultured in either control media or media treated with Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), or Salmonella typhimurium (S. typh) LPS. At 0 and 24 hours supernatants were assayed for prostaglandin-E2 (PGE2) and interleukin-1 beta (Il-1 beta) release by ELISA. Using flow cytometry the density of specific monocyte surface receptors were assayed with Mo3e and LeuM3 monoclonal antibodies (mAb); T-cell CD4/CD8 ratios were assayed with OKT-3, OKT-4, and OKT-8 mAb. After 24 hours incubation with Pg or S. typh LPS, the upregulation of the Mo3e receptor was significantly decreased for refractory periodontitis patients (P < 0.05) when compared to gingivitis and to stable maintenance patients. In refractory periodontitis patients the T-cell CD4/CD8 ratio was decreased. Upon stimulation with Pg or S. typh LPS, monocytes from stable maintenance and refractory periodontitis patients released more Il-1 beta (P < 0.05) and PGE2 (P = 0.13 and 0.15) than monocytes from gingivitis subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult

Tamoxifen as an antifertility agent in the long-tailed macaque (Macaca fascicularis).

The potential use of tamoxifen, a nonsteroidal antiestrogen, as an antifertility agent was studied in the long-tailed macaque (Macaca fascicularis). Twenty-six cycling females were bred, then treated with a single oral dose of tamoxifen (5 mg/kg) (N = 13) or vehicle (N = 13) on day 4 post-ovulation. Serum progesterone (P4) and tamoxifen concentrations were evaluated on post-ovulation days 4, 8, 12, 16, and 18. No effects of treatment were observed on P4 concentrations or on the fertility rate--pregnancy was achieved in 4/13 controls (31%) and 6/13 treated females (46%). Analysis for serum tamoxifen concentrations in samples collected during the fertility and a supplemental pharmacokinetic study (N = 3; single oral dose of 10 mg/kg; urine and serum evaluated) failed to reveal any detectable tamoxifen levels. It was concluded that (1) absorption of tamoxifen may be negligible under the described treatment regimens or (2) tamoxifen metabolism/clearance occurs at a rapid rate.

Administration, Oral

[Enhanced lymphocyte proliferation in the presence of epidermal cells of HIV-infected patients in vitro].

Clinical observations show that the HIV infection is often associated with affections of the skin. In order to examine the involvement of the epidermal immune system in the HIV infection, we determined accessory cell function of epidermal cells from HIV-1-infected patients. For this we measured the proliferative response of enriched CD(4+)-T-lymphocytes from HIV-infected patients and noninfected controls to stimulation with anti-CD3 and IL-2 in the presence of epidermal cells; the enhancement of the response is dependent on the presence of functionally intact accessory cells. The capacity of epidermal cells to increase the anti-CD3-stimulated T-cell proliferative response was significantly enhanced in HIV patients (CDC III/IVA) as compared with noninfected donors. It is discussed, whether the increased activity of epidermal cells from HIV-infected patients may be responsible for several of the dermal lesions in the course of an HIV infection as due to an enhanced production and release of epidermal cell-derived cytokines.

Adult

Single-cell cDNA-PCR: removal of contaminating genomic DNA from total RNA using immobilized DNase I.

A procedure utilizing immobilized DNase I that allows the efficient amplification of cDNA by PCR from a single cell in the absence of contaminating genomic DNA is described. DNase I treated, total RNA derived from single cells was reverse transcribed into cDNA followed by PCR using beta-actin and c-fos specific primers that recognize different exons of the respective genes. Amplification products corresponding to cDNA, but not to genomic sequences, were detected after treatment with immobilized DNase I in samples previously shown to be contaminated with genomic DNA. This method allows the efficient removal of DNA contaminating total RNA derived from a single cell.

Artifacts

Induction of high level IL-2 production in CD4+8- T helper lymphocytes requires post-thymic development.

Triggering of the CD3:TCR complex by optimal concentrations of anti-CD3, anti-TCR beta-chain, and allogeneic stimulator cells induced dramatically higher levels (fivefold for anti-CD3, greater than 10-fold for anti-TCR beta-chain, 84-fold for alloantigen) of IL-2 production in spleen CD4+8- T cells than their thymic counterparts, despite comparable levels of CD3 and TCR beta-chain expression. The nature of the reduced IL-2 production was examined by analysis of anti-CD3-induced IL-2 production at the single cell level. The frequency of IL-2-producing cells in spleen CD4+8- T cells (40.0%) was approximately threefold that of thymus CD4+8- T cells (14.5%). Furthermore, the average IL-2 levels among positive IL-2 producers was also approximately threefold higher in spleen CD4+8- T cells than their thymic counterparts. Adoptive transfer of purified Thy-1.2+ CD4+8- T cells into Thy-1.1-congenic hosts provided a physiologic and histocompatible system that enabled identification of transferred donor (Thy-1.2+) among a sea of host (Thy-1.2-) CD4+ T cells, whose immune function with respect to IL-2 inducibility was examined after isolation by electronic cell sorting. Donor CD4+ T cells thus isolated from host spleen shortly (1 day) after i.v. transfer of thymus CD4+8- T cells were similar to freshly isolated thymus CD4+8- T cells in that they both produced little IL-2 in response to anti-CD3. However, by day 3 post-transfer, IL-2 production by donor CD4+8- T cells had more than doubled and by day 8, they produced IL-2 levels comparable to those of host spleen CD4+8- T cells. A similar acquisition of high level IL-2 inducibility in thymus CD4+8- T cells upon i.v. transfer into Thy-1.1-congenic hosts was also observed using allogeneic cells as the stimulus of IL-2 production. When thymus CD4+8- T cells were intra-thymically transferred into Thy-1.1-congenic hosts, those donor cells that emigrated to the periphery became high IL-2 producers in a time-dependent manner, whereas those that remained inside the thymus showed no signs of up-regulation in IL-2 inducibility. Intrathymic transfer of CD4-8- thymocytes revealed that the most recent thymic emigrant CD4+8- T cells contained few IL-2-producing cells and were not functionally mature with respect to high level IL-2 inducibility.

Animals

Mitogen-induced IL-2 production and proliferation at defined stages of T helper cell development.

Th cell development inside the thymus can be defined on the basis of qualitative and quantitative CD4 and CD8 marker expression and follows the pathway of CD4-8- cells----CD4+8+ cells----CD4+8low cells----CD4+8- cells, which presumably emigrate to seed the periphery and serve as functionally mature Th cells. The various cell subpopulations at defined developmental stages were isolated by electronic cell sorting and examined for mitogen induced IL-2 production and cell proliferation responses. For TCR-alpha beta-bearing CD4+8+ and CD4+8low thymocytes that are actively engaged in positive and negative selection processes, negligible to low levels of IL-2 production and cell proliferation were observed in response to TCR:CD3 triggering or to the combined activation of protein kinase C and calcium mobilization mediated by PMA and ionomycin, respectively. For CD4-8- TCR-alpha beta early thymocytes that have not yet entered the selection process, PMA + ionomycin induced significant cell proliferation but little IL-2 production, in the absence of added IL-1. However, addition of IL-1 caused a powerful induction of IL-2 production that was accompanied by increased cell proliferation. Triggering of the TCR:CD3 complex had no effect on CD4-8-TCR(-)-alpha beta thymocytes as they do not express detectable levels of TCR-alpha beta. For thymus CD4+8- Th cells, the first cells that have completed TCR repertoire selection, vigorous proliferation was observed in response to TCR:CD3 triggering in the presence of added IL-2. However, the development of IL-2 responsiveness was not accompanied by high level IL-2 inducibility as TCR:CD3 triggering caused only marginal IL-2 production. In contrast, spleen CD4+8- T cells, the most "mature" representatives of Th cells, expressed high levels of IL-2 production as well as IL-2 responsiveness in response to TCR:CD3-mediated stimulation. The lack of anti-TCR-induced IL-2 production by thymus CD4+8- T cells was not due to an intrinsic defect as high levels of IL-2 production was induced by PMA + ionomycin. Possible reasons for the temporal acquisition and differential control of IL-2 inducibility and IL-2 responsiveness are discussed in the context of established Th cell development pathway.

Animals

The coherence of synthetic telomeres.

The chromosomal telomeres of Oxytricha were synthesized and their ability to cohere examined on non-denaturing acrylamide gels containing the stabilizing cation K+. At least 5 different mobility species were observed, in addition to that of the monomeric telomere. By cohering synthetic telomeres containing different lengths of subtelomeric DNA, we showed that each of the different mobility species was a dimer of two telomeres. Since the different mobility species did not differ in numbers or sequences of nucleotides, they must correspond to different molecular shapes probably caused by different degrees of bending of the dimer. Paradoxically, telomeres with longer subtelomeric stems cohered more efficiently. In the presence of K+, solutions had to be heated to over 90 degrees before the telomeres separated. Various synthetic constructs, restriction endonuclease and dimethyl sulfate protection experiments showed that the only nucleotides involved in the cohered structures were the 16 base 'tails' of sequence 3'G4T4G4T4. Extension of this motif was actually inimical to coherence. Oligomers containing 2 G4T4 motifs protected their GN7 positions by forming dimers, those with 5 G4T4 could do so by internal folding, but the 3' terminal group of G4 was left unprotected. This suggests that only four groups of G4 are necessary for the cohered structure. Single-chain specific nuclease, S1, as well as osmium tetroxide, which oxidizes the thymine residues of single chains, reacted less efficiently with the cohered structures. Synthetic telomeres containing inosine replacing guanosine were not observed to cohere, indicating that the C2-NH2 is strongly stabilizing. The cohered structures appear to be unusually compact and sturdy units in which four G4 blocks form quadruplexes stabilized by K+. A new model for the cohered structure is presented.

Animals

Subpopulations of CD8+ cytotoxic T cell precursors collaborate in the absence of conventional CD4+ helper T cells.

Four different subpopulations (Ly6Cneg, Ly6Clow, Ly6Cint, and Ly6Chi) of CD8+ T cells were arbitrarily defined on the basis of differential expression of Ly6C Ag. By combining the processes of electronic cell sorting and automated cell deposition, small numbers of respective CD8+ T cell subpopulations were directly deposited into tissue culture wells in which mitogen-stimulated responses were studied. Anti-CD3-stimulated proliferation and IL-2 production were the strongest by Ly6Cneg/Ly6Clow T cells, moderate for Ly6Cint T cells, and highly deficient for Ly6Chi T cells. The level of IL-2 production for Ly6Cneg CD8+ T cells was comparable to that of conventional CD4+ Th cells. Allogeneic stimulator cells elicited a strong cytotoxic response by Ly6Cneg + low but not Ly6Chi CD8+ T cells in the absence of added lymphokines. When IL-2 was supplied in excess, anti-CD3 induced comparable levels of cell proliferation and cytotoxic activity in Ly6Cneg, Ly6Clow, Ly6Cint, and Ly6Chi CD8+ T cells whereas alloantigen stimulated an approximate fivefold higher cytotoxic response by Ly6Chi than Ly6Cneg + low CD8+ T cells. Stimulation of co-cultures of B10 (CD8b) Ly6Cneg + low and congenic B10.CD8a Ly6Chi CD8+ T cells in the absence of added lymphokines, followed by selective elimination of activated CD8.1+ (CD8.2+) T cells by anti-CD8.1 (anti-CD8.2) + C treatment, allowed the demonstration that help provided by Ly6Cneg + low T cells can be effectively used by both Ly6Cneg + low and Ly6Chi T cells in anti-CD3 and alloantigen induced proliferative and cytotoxic responses, respectively.

Animals

Bactericidal activity of human peritoneal macrophages from patients undergoing peritoneal dialysis.

Peritoneal macrophages (PM) play an essential role in the pathogenesis of bacterial peritonitis, the main complication of peritoneal dialysis (PD). We determined the antibacterial activity of PM from 31 PD patients using gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and gram-negative (Escherichia coli, Pseudomonas aeruginosa) test organisms. In an 8-hour test assay, PM revealed the highest antibacterial activity against E. coli [median bactericidal index (Bi) = 5.46 representing 0.74 log growth inhibition compared to controls] and the lowest against P. aeruginosa (Bi = 1.63, 0.21 log growth inhibition, p less than 0.05). The antibacterial activity against S. aureus (Bi = 1.99, 0.3 log growth inhibition) and S. epidermidis (Bi = 2.0, 0.31 log growth inhibition) was within this range. When compared to peripheral blood polymorphonuclear leukocytes, PM reached only 4% (S. aureus) and 8.1% (E. coli) of their antibacterial activity (p less than 0.05). Using E. coli as a test organism, PM isolated after a 4-hour dialysis period revealed the highest antibacterial activity when compared to PM isolated after longer dialysis periods (p less than 0.05). Increasing the duration of PD to 6 and 8 h subsequently decreased the antibacterial activity of PM, suggesting that unphysiologic concentrations of toxic metabolites in the peritoneal effluent might have a harmful influence on PM functions.

Bacteria