Effect of various factors on the biosynthesis of piscarinines,secondary metabolites of the fungus <Emphasis Type="Italic">Penicillium piscarium</Emphasis> Westling

Piscarinines A and B were synthesized most actively during the surface cultivation of the fungus Penicillium piscarium in a complex medium (5.5 mg/l). Under conditions of submerged cultivation in a mineral medium, the yield of piscarinines was two times lower. An increase in the inoculum quantity of conidia treated with Tween-80 increased the culture productivity. The biosynthesis of the alkaloid was completely suppressed when mannitol was replaced with glucose or when zinc, iron, or copper ions were added to the culture medium. The metabolites were active against the prostate cancer cell line LNCAP (IC₅₀ were 2.195 and 1.914 μg/ml for piscarinines A and B, respectively).     

Reversal of the Mannitol-Sorbitol Diauxie in Escherichia coli

In Escherichia coli K-12 the proteins involved in the dissimilation of mannitol and sorbitol are specified by two separate gene clusters. The mannitol cluster appears to consist of a regulatory gene mtlC, a gene mtlA coding an enzyme II complex of the phosphoenolpyruvate phosphotransferase system, and a gene mtlD coding a mannitol-1-phosphate dehydrogenase. Three corresponding genes, sblC, sblA, and sblD, exist for the sorbitol pathway. In both pathways the hexitol captured from the medium and delivered into the cytoplasm as a phosphorylated compound is dehydrogenated to fructose-6-phosphate. The enzyme II complex for sorbitol is able to catalyze the phosphorylation also of mannitol if this substrate is present at high concentrations. Consequently mtlA(-) mutants lacking the enzyme II complex for mannitol can grow on mannitol either if the sorbitol phosphorylating system is preinduced by sorbitol or if mtlA is suppressed by a mutation of sblC to constitutivity. In wild-type cells, the induction of the enzymes in the mannitol pathway and dissimilation of the substrate are not prevented by glucose. The sorbitol system, however, is sensitive to glucose and to mannitol as well. In the suppressed strains (mtlA(-), sblC(c)) in which mannitol is utilized through the sorbitol enzyme, glucose becomes effective in restraining the consumption of mannitol, causing a definite diauxie. Moreover, in a mixture of mannitol and sorbitol, the latter is utilized preferentially. This reversal of normal diauxic pattern is consequent to the fact that the enzyme II complex for sorbitol has relatively poor affinity for mannitol.     

Effect of the cultivation conditions on alkaloid biosynthesis by Penicillium gorlenkoanum

The effect of a carbohydrate component of the medium, trace elements and aeration on biosynthesis of the alkaloids costaclavine and epicostaclavine was studied with Penicillium gorlenkoanum. Alkaloid biosynthesis was shown to depend on the nature of a carbohydrate component: virtually no alkaloids were accumulated in media with glucose and fructose although these were synthesized at a high rate in a medium with mannitol. The quantity of synthesized alkaloids and the dynamics of the biosynthesis depended on carbohydrate concentration. The growth and alkaloid synthesis were influenced by traces of zinc, iron, copper and manganese. A more intensive aeration stimulated biomass accumulation but suppressed alkaloid biosynthesis.     

Glucose and nutrient concentrations affect the expression of a 104-kilodalton Listeria adhesion protein in Listeria monocytogenes

Growth media and environmental conditions influence the expression of adhesion and invasion proteins in Listeria monocytogenes. Here, the expression of the 104-kDa Listeria adhesion protein (LAP) was studied in nutrient-rich media (Trypticase soy broth [TSB] and brain heart infusion [BHI]), minimal medium (Luria-Bertani [LB]), or nutrient-deficient medium (peptone water [PW]) by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Also, the effect of incorporating different concentrations of glucose on LAP expression was studied. Immunoblotting showed that LAP expression was at least twofold higher in LB medium than in TSB or BHI, while PW supported very poor cell growth and LAP expression. ELISA and immunoblotting results showed that higher concentrations of glucose (>1.6 g/liter) lowered the culture pH and suppressed LAP expression by more than 75%; however, the addition of K(2)HPO(4) reduced this effect. L. monocytogenes cells grown in LB media with lower concentrations of glucose showed higher adhesion to Caco-2 cells (3,716 and 4,186 cpm of attached bacteria for 0 and 0.2 g of glucose/liter, respectively), while L. monocytogenes cells grown in LB with higher glucose concentrations exhibited lower adhesion (2,126 and 2,221 cpm for 1.6 and 3.2 g of glucose/liter, respectively). A LAP-negative L. monocytogenes strain (A572) showed low adhesion profiles regardless of the amount of glucose added. Transmission electron microscopy revealed that LAP is localized mainly in the cytoplasm, with only a few molecules located on the cell surface. Growth in LB with high glucose (3.2 g/liter) showed the presence of only a few molecules in the cells, corroborating the results observed with ELISA or immunoblotting. In summary, nutrient-rich media and high concentrations of glucose suppressed LAP expression, which possibly is due to the changes in the pH of the media during growth from the accumulation of sugar fermentation by-products.     

Creation of cell surface-engineered yeast that display different fluorescent proteins in response to the glucose concentration

We have successfully created a novel yeast strain able to monitor changes in environmental conditions by displaying either green fluorescent protein (GFP) from Aequorea victoria or blue fluorescent protein (BFP), a variant of GFP, on its cell surface as a visible reporter. For the display of these fluorescent proteins on the cell surface of Saccharomyces cerevisiase, our cell-surface-engineering system was utilized. The GAPDH promoter, which is active in the presence of glucose, and the UPR-ICL promoter from Candida tropicalis, which starts to function in the presence of a reduced level of glucose, were employed simultaneously to express the GFP-encoding gene and the BFP-encoding gene, respectively. This cell-surface-engineered yeast strain emitted green fluorescence from the cell surface when sufficient glucose was present in the medium, and blue fluorescence from the same cell surface when the glucose in the medium was consumed. The fluorescent proteins displayed on the cell surface using the different promoters enabled us to monitor the concentrations of intra- and/or extracellular glucose that regulated activation or inactivation of the promoters. This novel yeast strain could facilitate the computerized control of various bioprocesses measuring emitted fluorescence.     

Effect of mannitol and glucose-induced osmotic stress on growth,water relations,and solute composition of cell suspension cultures of poplar (Populus deltoides var.Occidentalis) in relation to anthocyanin accumulation

Summary A cell suspension culture of poplar (Populus deltoides (Marsh.) Bartr. var.occidentalis Rydb.), accumulating the anthocyanin pigment, cyanidin 3-glucoside, in the lag phase of culture growth, was subjected to osmotic stress with glucose and mannitol. Osmotic stress treatments resulted in growth suppression and higher anthocyanin accumulation compared with unstressed cells. Both an increase in the proportion of pigmented cells and an increase in the concentration of anthocyanin in the pigmented cells were responsible for high anthocyanin content of cultured cells subjected to osmotic stress. The osmotic stress induced by glucose suppressed growth more than that by mannitol and produced higher anthocyanin levels. Only small amounts of [U-¹⁴C]mannitol were taken up and metabolized by the cells. Stressed cells accumulated sugars and free amino acids to a different extent resulting in altered cell sugar-to-amino acid ratios. The accumulation of osmotically active solutes and cell growth suppression may both be responsible for the accumulation of anthocyanin in stressed cells.     

Glucose and Nutrient Concentrations Affect the Expression of a 104-Kilodalton Listeria Adhesion Protein in Listeria monocytogenes

Growth media and environmental conditions influence the expression of adhesion and invasion proteins in Listeria monocytogenes. Here, the expression of the 104-kDa Listeria adhesion protein (LAP) was studied in nutrient-rich media (Trypticase soy broth [TSB] and brain heart infusion [BHI]), minimal medium (Luria-Bertani [LB]), or nutrient-deficient medium (peptone water [PW]) by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Also, the effect of incorporating different concentrations of glucose on LAP expression was studied. Immunoblotting showed that LAP expression was at least twofold higher in LB medium than in TSB or BHI, while PW supported very poor cell growth and LAP expression. ELISA and immunoblotting results showed that higher concentrations of glucose (>1.6 g/liter) lowered the culture pH and suppressed LAP expression by more than 75%; however, the addition of K₂HPO₄ reduced this effect. L. monocytogenes cells grown in LB media with lower concentrations of glucose showed higher adhesion to Caco-2 cells (3,716 and 4,186 cpm of attached bacteria for 0 and 0.2 g of glucose/liter, respectively), while L. monocytogenes cells grown in LB with higher glucose concentrations exhibited lower adhesion (2,126 and 2,221 cpm for 1.6 and 3.2 g of glucose/liter, respectively). A LAP-negative L. monocytogenes strain (A572) showed low adhesion profiles regardless of the amount of glucose added. Transmission electron microscopy revealed that LAP is localized mainly in the cytoplasm, with only a few molecules located on the cell surface. Growth in LB with high glucose (3.2 g/liter) showed the presence of only a few molecules in the cells, corroborating the results observed with ELISA or immunoblotting. In summary, nutrient-rich media and high concentrations of glucose suppressed LAP expression, which possibly is due to the changes in the pH of the media during growth from the accumulation of sugar fermentation by-products.     

Osservazioni Sull'effetto Inibente di Elevate Pressioni Osmotiche su Diversi Tipi di Crescita

Abstract

On the inhibition of seeds germination and of growth by cell enlargement by the osmotic pressure of the medium. — The mechanism of inhibition by osmotic pressure (O.P.) of the medium on growth and respiration of germinating wheat, castor bean and lettuce sèeds and of etiolated pea internode segments was investigated.

The following results were obtained:

1 - External osmotic pressure (up to 0.3 M) of various substances such as mannitol, urea, glucose, NaCl, was shown to inhibit the germination and growth of lettuce, wheat and castor bean seeds.

2 - a) A remarkable decrease of the development of respiration during the first 48 h of germination was demonstrated in embryos of wheat seeds germinated and maintained in mannitol solutions at concentration from 0,2 to 0,3 M.

b) A slight but reproducible inhibition of óxygen uptake by O.P. was also observed in embryos isolated from wheat seeds germinated in water for 24 and 34 h and transported respectively in water or into 0,2 M mannitol solutions.

This is interpreted as indicating that high external O.P. inhibits both the respiratory metabolism and the development with time of enzyme systems supporting respiration.

3 - Mannitol solutions (0,2–0,3 M) inhibited completely growth by cell enlargement in pea internode sections, while they did not at all affect oxygen uptake and protein synthesis ( ¹⁴ C - leucine incorporation). The stimulatory effect of auxin on pea elongation was almost completely suppressed by mannitol, whereas the hormone stimulation of respiration remained unchanged.

These data are interpreted as indicating that in tissues, presenting an advanced differentiation, high external O.P. inhibits growth by a direct physico-chemical mechanism; while the inhibitory effect in embrional tissues seems to comprehend, besides this direct effect, a complicated metabolic component, apparently influencing protein synthesis.     

Some effects of mannitol on the glucose metabolism of two derivatives of a strain ofRhizobium japonicum differing in mannitol dehydrogenase

The effects of mannitol were investigated by comparing some metabolic features in colonial derivatives, I-110 and L1-110, ofRhizobium japonicum strain 3IIb110, grown either on glucose alone (G-cells) or in glucose media supplemented with mannitol (GM-cells). The polyol stimulated the synthesis of not only mannitol dehydrogenase, which is active in derivative L1-110, but also the nicotinamide adenine dinucleotide (NAD)-linked 6-phosphogluconate (6-PG) dehydrogenase (EC 1.1.1.43). As revealed by radiorespirometry, when GM-cells were allowed to metabolize glucose, they produced relatively more CO₂ from the first and sixth carbons of the sugar than G-cells did. This finding is evidence that NAD-linked 6-PG dehydrogenase might initiate an unknown pathway different from the hexose cycle and the pentose phosphate (PP) pathway. Mannitol exerted no allosteric control on the oxygen consumption and the glucose transport systems. Active uptake of the polyol was correlated with the presence of mannitol dehydrogenase (EC 1.1.1.67); it did not hinder the transport of glucose even though both systems derive their energy for active transport from a common source presumptively characterized as the energized membrane state. Mannitol, however, suppressed by two- or threefold the glucose uptake system. Addition of the polyol to the cell suspensions of both colonial types ofR. japonicum metabolizing glucose caused an immediate 40–50% drop of adenosine triphosphate (ATP) concentrations, owing in part to the mannitol kinase reaction. Type I-110 failed to overcome this reduction of ATP levels, and low growth rates could results. In contrast, type L1-110 offsets the reduction of ATP concentration by oxidizing mannitol as an additional source of energy through mannitol dehydrogenase, fructokinase, and a sequence of glycolytic reactions. The polyol also induced type L1-110 to produce extracellular slimy materials that, apparently, harbor amounts of ATP and proteins.     

Temporal accumulation of mannitol and arabitol in Geotrichum candidum

The temporal depletion and accumulation of polyols were investigated in the fungus Geotrichum candidum. The major intracellular polyols were tentatively identified by paper chromatography as mannitol and arabitol. Inositol was also present in small quantities, and trehalose was also detected in appreciable concentrations.Germination and vegetative growth depended on the type and concentration of the sole exogenous carbon source. Mannitol occurred in arthrospores at 9.4% of the dry weight after several days growth in 2% (w/v) glucose solid medium, and became depleted during germination and vegetative growth in liquid medium containing 2% (w/v) glucose, 2% (w/v) sodium acetate or 25% (w/v) glucose as sole carbon source. This hexitol latter accumulated during arthrosporulation. The depletion and accumulation of ethanol-soluble carbohydrate believed to be primarily trehalose was temporally similar to that of mannitol. Arabitol accumulated intracellularly during germination and vegetative growth in sodium acetate medium and 25% glucose medium. This pentitol was not detected intracellularly at any culture age during growth in 2% glucose medium.Prolonged incubation of the culture in 25% glucose medium after stationary phase was reached resulted in the gradual disappearance of arabitol from the arthrospores simultaneously with an increase in intracellular mannitol. In comparison, ethanol-soluble carbohydrate did not change with prolonged incubation in this medium.     

Isolation and expression of an elongation-dependent gene of mung bean (Vigna radiata) hypocotyl

A cDNA clone of an auxin up-regulated gene, ARG8 , was isolated from hypocotyl sections of etiolated mung bean [ Vigna radiata (L.) Wilczek] seedlings by differential screening. The deduced amino acid sequence suggested that ARG8 may encode a cell wall protein. The steady state mRNA level of ARG8 increased by treatment of hypocotyl sections not only with indole-3-acetic acid (IAA) but also with fusicoccin, and the auxin inducibility was inhibited by the addition of 0.3 M mannitol in the incubation medium. This indicated that it was not auxin but elongation that regulated the expression of ARG8 . The promoter activity of the 5'-flanking region of ARG8 was determined by assaying the transient expression of a luciferase fusion gene that was introduced into mung bean hypocotyl sections by the particle bombardment technique. The basal activity of the ARG8 upstream region was about a few tenths of that of a modified cauliflower mosaic virus 35S promoter, and it was increased a few fold by treatment with IAA. The auxin inducibility was completely suppressed by the addition of mannitol. A 5'-deletion analysis showed that a 53-bp region in the ARG8 promoter was important for the basal and elongation-dependent promoter activities.     

Glucose metabolism in Neurospora is altered by heat shock and by disruption of HSP30

We compared the metabolism of [1-13C]glucose by wild type cells of Neurospora crassa at normal growth temperature and at heat shock temperatures, using nuclear magnetic resonance analysis of cell extracts. High temperature led to increased incorporation of 13C into trehalose, relative to all other metabolites, and there was undetectable synthesis of glycerol, which was a prominent metabolite of glucose at normal temperature (30 degrees C). Heat shock strongly reduced formation of tricarboxylic acid cycle intermediates, approximately 10-fold, and mannitol synthesis was severely depressed at 46 degrees C, but only moderately reduced at 45 degrees C. A mutant strain of N. crassa that lacks the small alpha-crystallin-related heat shock protein, Hsp30, shows poor survival during heat shock on a nutrient medium with restricted glucose. An analysis of glucose metabolism of this strain showed that, unlike the wild type strain, Hsp30-deficient cells may accumulate unphosphorylated glucose at high temperature. This suggestion that glucose-phosphorylating hexokinase activity might be depressed in mutant cells led us to compare hexokinase activity in the two strains at high temperature. Hexokinase was reduced more than 35% in the mutant cell extracts, relative to wild type extracts. alpha-Crystallin and an Hsp30-enriched preparation protected purified hexokinase from thermal inactivation in vitro, supporting the proposal that Hsp30 may directly stabilize hexokinase in vivo during heat shock.     

Plant Regeneration from Callus Protoplasts of Codonopsis pilosula

Embryogenic cell line was established from hypocotyl segments of Codonopsis pilosula (Franch.)Nannf. 4--8 day old embryogenic callus was used to isolate protoplasts in an enzyme solution containing 1.5 % cellulase Onozuka R-10 and 3 % pectinase. Protoplasts were cultured in MS,C81V,DPD and KMSp basal medium supplemented with 1.2 mg/L 2,4-D, 0.2 mg/L NAA, 0. 2 mg/L BAP, 0. 1 mg/L ZT,and different combinations of glucose and mannitol . Protoplast-derived cells underwent sustained divisions in KM8p medium. As an osmoticum, glucose was more beneficial to protoplast division. A combination of 0. 30 mol/L glucose with 0.10 mol/L mannitol gave the best result. Under proper conditions , protoplasts underwent the first division on the 3rd day of culture,formed colonies within 30 days , and developed into microcalli in 6 weeks. Plantlets were regenerated from protoplast-derived calli through somatic embryogenesis. 0.2 % activated charcoal promoted embryoid formation and root development.     

Dependence of activities of polysaccharide hydrolases and oxidases from Cerrena unicolor on the source of carbon and aromatic acids in culture media

The activities of carboxymethylcellulase and xylanase in the higher basidial fungus Cerrena unicolor grown in avicel-containing medium reached 1.95 and 1.50 units per mg protein, respectively, whereas in mannitol-containing medium they ranged from 0.02 to 0.05 units per mg protein. The activity of fungal beta-glucosidase depended on the carbon source in the culture medium and ranged from 2.1 units per mg protein in the presence of mannitol to 17.3 units per mg protein in the presence of avicel. In contrast to polysaccharides, easily metabolizable substrates (cellobiose, mannitol, and glucose) provided the highest rates of secretion of laccase (52.7-123.5 ncat per mg protein) and ligninase (22-106 units per mg protein). The addition of tangerine pomace, a substrate enriched with aromatic compounds, to the culture medium caused an increase in the rate of bio-synthesis of laccase and ligninase to 862 ncat per ml and 557 units per ml, respectively. Aromatic compounds such as p-xylidine and veratric aldehyde increased the laccase activity of C. unicolor IBB 62 from 7.9 to 23.6 and 18.3 ncat per mg protein, respectively. Veratryl alcohol caused a sevenfold increase in the activity of Mn-dependent peroxidase in the culture medium.     

Insertion of the mannitol permease into the membrane of Escherichia coli. Possible involvement of an N-terminal amphiphilic sequence

The in vivo membrane assembly of the mannitol permease, the mannitol Enzyme II (IImtl) of the Escherichia coli phosphotransferase system, has been studied employing molecular genetic approaches. Removal of the N-terminal amphiphilic leader of the permease and replacement with a short hydrophobic sequence resulted in an inactive protein unable to transport mannitol into the cell or catalyze either phosphoenol-pyruvate-dependent or mannitol 1-phosphate-dependent mannitol phosphorylation in vitro. The altered protein (68 kDa) was quantitatively cleaved by an endogenous protease to a membrane-associated 39-kDa fragment and a soluble 28-kDa fragment as revealed by Western blot analyses. Overproduction of the wild-type plasmid-encoded protein also led to cleavage, but repression of the synthesis of the plasmid-encoded enzyme by inclusion of glucose in the growth medium prevented cleavage. Several mtlA-phoA gene fusions encoding fused proteins with N-terminal regions derived from the mannitol permease and C-terminal regions derived from the mature portion of alkaline phosphatase were constructed. In the first fusion protein, F13, the N-terminal 13-aminoacyl residue amphiphilic leader sequence of the mannitol permease replaced the hydrophobic leader sequence of alkaline phosphatase. The resultant fusion protein was inefficiently translocated across the cytoplasmic membrane and became peripherally associated with both the inner and outer membranes, presumably via the noncleavable N-terminal amphiphilic sequence. The second fusion protein, F53, in which the N-terminal 53 residues of the mannitol permease were fused to alkaline phosphatase, was efficiently translocated across the cytoplasmic membrane and was largely found anchored to the inner membrane with the catalytic domain of alkaline phosphatase facing the periplasm. This 53-aminoacyl residue sequence included the amphiphilic leader sequence and a single hydrophobic, potentially transmembrane, segment. Analyses of other MtlA-PhoA fusion proteins led to the suggestion that internal amphiphilic segments may function to facilitate initiation of polypeptide trans-membrane translocation. The dependence of IImtl insertion on the N-terminal amphiphilic leader sequence was substantiated employing site-specific mutagenesis. The N-terminal sequence of the native permease is Met-Ser-Ser-Asp-Ile-Lys-Ile-Lys-Val-Gln-Ser-Phe-Gly.... The following point mutants were isolated, sequenced, and examined regarding the effects of the mutations on insertion of IImtl into the membrane: 1) S3P; 2) D4P; 3) D4L; 4) D4R; 5) D4H; 6) I5N; 7) K6P; and 8) K8P.(ABSTRACT TRUNCATED AT 400 WORDS)     

NKCC activity restores muscle water during hyperosmotic challenge independent of insulin,ERK, and p38 MAPK

In isosmotic conditions, insulin stimulation of PI 3-K/Akt and p38 MAPK pathways in skeletal muscle inhibits Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity induced by the ERK1,2 MAPK pathway. Whether these signaling cascades contribute to NKCC regulation during osmotic challenge is unknown. Increasing osmolarity by 20 mosM with either glucose or mannitol induced NKCC-mediated (86)Rb uptake and water transport into rat soleus and plantaris skeletal muscle in vitro. This NKCC activity restored intracellular water. In contrast to mannitol, hyperosmolar glucose increased ERK1,2 and p38 MAPK phosphorylation. Glucose, but not mannitol, impaired insulin-stimulated phosphorylation of Akt and p38 MAPK in the plantaris and soleus muscles, respectively. Hyperosmolarity-induced NKCC activation was insensitive to insulin action and pharmacological inhibition of ERK1,2 and p38 MAPK pathways. Paradoxically, cAMP-producing agents, which stimulate NKCC activity in isosmotic conditions, suppressed hyperosmolar glucose- and mannitol-induced NKCC activity and prevented restoration of muscle cell volume in hyperosmotic media. These results indicate that NKCC activity helps restore muscle cell volume during hyperglycemia. Moreover, hyperosmolarity activates NKCC regulatory pathways that are insensitive to insulin inhibition.     

The structure of a mannitol teichoic acid from Bifidobacterium bifidum ssp. Pennsylvanicum

The isolation and analysis of the cell wall and cell wall fractions of Bifidobacterium bifidum ssp. pennsylvanicum are presented. With lysozyme a solubilized cell wall fraction is obtained which contains muramic acid, glucosamine, rhamnose, glucose, mannitol, phosphate and all peptidoglycan amino acids. Its composition did not change with culture age. A glycogen-like glucose polymer which is of cytoplasmic origin is identified in the insoluble cell wall fraction. The solubilized cell wall fraction contains a glucosylated rhamnose polymer which is linked by glycosidic bonds to the peptidoglycan fragments. This polymer is a 1,2-linked or an alternating, 1,2/1,3-linked α-rhamnose chain substituted on average at every second rhamnose residue with an α-linked glucose molecule. Various experiments gave evidence that mannitol and phosphate are present in 4,6-linked mannitol phosphate oligomers which are linked by phosphodiester bonds to the glucosylated rhamnose polymer. These oligomers may fulfill the functions of the more common wall teichoic acids.     

Attenuation of mouse mesangial cell contractility by high glucose and mannitol: Involvement of protein kinase C and focal adhesion kinase

Hyperglycemia and mannitol activate protein kinase C (PKC) and induce mesangial cell hypocontractility that subsequently may modulate renal function. Since focal adhesion kinase (FAK) activation is known to be linked with PKC activity, FAK may also be involved in mesangial cell contraction. To facilitate our understanding of the PKC- and FAK-modulating mechanism, we developed an in vitro model of mouse mesangial cell hypocontractility induced by hyperglycemia or mannitol. Mouse mesangial cells (CRL-1927) were exposed to: normal D-glucose (group N), high D-glucose (group H), and control groups at the same osmolality as H plus L-glucose (group L) and mannitol (group M). Changes in the planar surface area of cells in response to 1 microM phorbol 12-myristate 13-acetate (PMA) were determined. Western blot analyses for PKC, phosphorylated (p)-PKC, tyrosine phosphorylation, FAK, and p-FAK were done on each of these four groups. The effects of mannitol in various doses on cell contraction and activation of PKC and FAK were also assayed. The planar surface areas of groups H and M both showed an attenuated change in response to PMA stimulation. Before PMA stimulation, the baseline PKC expression of groups H and M showed a higher expression of p-PKC alpha and delta than that seen in group N (p < 0.05). Results of tyrosine phosphorylation and immunoprecipitation showed that FAK may be involved in this contraction process. The total amount of FAK showed no significant difference among the four experimental groups; however, p-FAK was found to have significantly increased in group M (p < 0.05). The use of PKC and tyrosine kinase inhibitors reduced PMA-induced mesangial cell contraction in all four groups. Activation of PKC alpha, delta, and FAK with the resultant inhibition of mesangial cell contraction by mannitol was found to be dose-dependent. These results may provide a correlation between increased expression of several PKC isoforms and, in particular, increased phosphorylation levels of PKC alpha and delta and hypocontractility induced by high glucose and mannitol treatment. Furthermore, the mannitol-induced hypocontractility involving PKC and FAK occurred in a dose-dependent manner.