Presence Of Growth Medium Research Articles

In Vitro Biocompatibility Assessment of Bioengineered PLA-Hydrogel Core-Shell Scaffolds with Mesenchymal Stromal Cells for Bone Regeneration.

Human mesenchymal stromal cells (hMSCs), whether used alone or together with three-dimensional scaffolds, are the best-studied postnatal stem cells in regenerative medicine. In this study, innovative composite scaffolds consisting of a core-shell architecture were seeded with bone-marrow-derived hMSCs (BM-hMSCs) and tested for their biocompatibility and remarkable capacity to promote and support bone regeneration and mineralization. The scaffolds were prepared by grafting three different amounts of gelatin-chitosan (CH) hydrogel into a 3D-printed polylactic acid (PLA) core (PLA-CH), and the mechanical and degradation properties were analyzed. The BM-hMSCs were cultured in the scaffolds with the presence of growth medium (GM) or osteogenic medium (OM) with differentiation stimuli in combination with fetal bovine serum (FBS) or human platelet lysate (hPL). The primary objective was to determine the viability, proliferation, morphology, and spreading capacity of BM-hMSCs within the scaffolds, thereby confirming their biocompatibility. Secondly, the BM-hMSCs were shown to differentiate into osteoblasts and to facilitate scaffold mineralization. This was evinced by a positive Von Kossa result, the modulation of differentiation markers (osteocalcin and osteopontin), an expression of a marker of extracellular matrix remodeling (bone morphogenetic protein-2), and collagen I. The results of the energy-dispersive X-ray analysis (EDS) clearly demonstrate the presence of calcium and phosphorus in the samples that were incubated in OM, in the presence of FBS and hPL, but not in GM. The chemical distribution maps of calcium and phosphorus indicate that these elements are co-localized in the same areas of the sections, demonstrating the formation of hydroxyapatite. In conclusion, our findings show that the combination of BM-hMSCs and PLA-CH, regardless of the amount of hydrogel content, in the presence of differentiation stimuli, can provide a construct with enhanced osteogenicity for clinically relevant bone regeneration.

A heparin-functionalized carbon nanotube-based affinity biosensor for dengue virus

Dengue virus is an arthropod-borne virus transmitted primarily by Aedes mosquitos and is major cause of disease in tropical and subtropical regions. Colloquially known as Dengue Fever, infection can cause hemorrhagic disorders and death in humans and non-human primates. We report a novel electronic biosensor based on a single-walled carbon nanotube network chemiresistive transducer that is functionalized with heparin for low-cost, label-free, ultra-sensitive, and rapid detection of whole dengue virus (DENV). Heparin, an analog of the heparan sulfate proteoglycans that are receptors for dengue virus during infection of Vero cells and hepatocytes, was used for the first time in a biosensor as a biorecognition element instead of traditional antibody. Detection of DENV in viral culture supernatant has similar sensitivity as the corresponding viral titer in phosphate buffer despite the presence of growth media and Vero cell lysate. The biosensor demonstrated sensitivity within the clinically relevant range for humans and infected Aedes aegypti. It has potential application in clinical diagnosis and can improve point-of-care diagnostics of dengue infection.

A new design for an artificial cell: polymer microcapsules with addressable inner compartments that can harbor biomolecules, colloids or microbial species.

Eukaryotic cells have an architecture consisting of multiple inner compartments (organelles) such as the nucleus, mitochondria, and lysosomes. Each organelle is surrounded by a distinct membrane and has unique internal contents; consequently, each organelle has a distinct function within the cell. In this study, we create biopolymer microcapsules having a compartmentalized architecture as in eukaryotic cells. To make these capsules, we present a biocompatible method that solely uses aqueous media (i.e., avoids the use of oil phases), requires no sacrificial templates, and employs a minimal number of steps. Our approach exploits the electrostatic complexation of oppositely charged polymers dissolved in aqueous media. Specifically, droplets of an anionic biopolymer are generated using a simple microcapillary device, with the droplets being sheared off the capillary tip by pulses of gas (air or nitrogen). The liquid droplets are then introduced into a reservoir whereupon they encounter multivalent cations as well as a cationic biopolymer; thereby, a solid shell is formed around each droplet by electrostatic interactions between the polymers while the core is ionically cross-linked into a gel. In the next step, a discrete number of these capsules are encapsulated within a larger outer capsule by repeating the same process with a wider capillary. Our approach allows us to control the overall diameter of these multicompartment capsules (MCCs) (∼300-500 μm), the diameters of the inner compartments (∼100-300 μm), and the number of inner compartments in an MCC (1 to >5). More importantly, we can encapsulate different payloads in each of the inner compartments, including colloidal particles, enzymes, and microbial cells, in all cases preserving their native functions. A hallmark of biological cells is the existence of cascade processes, where products created in one organelle are transported and used in another. As an initial demonstration of the capabilities afforded by our MCCs, we study a simple cascade process involving two strains of bacteria (E. coli), which communicate through small molecules known as autoinducers. In one compartment of the MCC, we cultivate E. coli that produces autoinducer 2 (AI-2) in the presence of growth media. The AI-2 then diffuses into an adjacent compartment within the MCC wherein a reporter strain of E. coli is cultivated. The reporter E. coli imbibes the AI-2 and in turn, produces a fluorescence response. Thus, the action (AI-2 production) and response (fluorescence signal) are localized within different compartments in the same MCC. We believe this study is an important advance in the path towards an artificial cell.

Effect of graphene oxide on bacteria and peripheral blood mononuclear cells.

Driven by the potential biological applications of graphene, many groups have studied the response of cells exposed to graphene oxide (GO). In particular, investigations of bacteria indicate that there are 2 crucial parameters, which so far have only been investigated separately: GO size and exposure methodology. Our study took into account both parameters. We carefully characterized the samples to catalog sizes and structural properties, and tested different exposure methodologies: exposure in saline solution and in the presence of growth media. Furthermore, we performed experiments with peripheral blood mononuclear cells exposed to our GO materials. Atomic force microscopy, scanning electron microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy and transmission electron microscopy were used to characterize the morphology and composition of different samples of GO: GO-H2O, GO-PBS and GO-MG. Our samples had 2D sizes of ~100 nm (GO-H2O and GO-PBS) and >2 µm (GO-MG). We tested antibacterial activity and cytotoxicity toward peripheral blood mononuclear cells of 3 different GO samples. A size-dependent growth inhibition of Escherichia coli (DH5 α) in suspension was found, which proved that this effect depends strongly on the protocol followed for exposure. Hemocompatibility was confirmed by exposing peripheral blood mononuclear cells to materials for 24 hours; viability and apoptosis tests were also carried out. Our experiments provide vital information for future applications of GO in suspension. If its antibacterial properties are to be potentiated, care should be taken to select 2D sizes in the micrometer range, and exposure should not be carried out in the presence of grow media.

Abstract P5-09-19: Progesterone receptor isoform A as a predictive marker of antiprogestin therapy in breast cancer: A tissue culture method to evaluate drug responsiveness

Abstract Two thirds of breast cancers express steroid hormone receptors and are susceptible to an endocrine therapy usually aimed to target the estrogen receptors (ER). There is however compelling evidence suggesting that progesterone receptors (PR) participate together with ER, modulating breast cancer growth. Based on our results obtained in an experimental breast cancer model in which antiprogestins induce complete regression of carcinomas with higher levels of PR isoform A (PR-A) than isoform B (PR-B), we propose that antiprogestins such as mifepristone (MFP) might be a possible therapeutic option for patients expressing high levels of PR-A. The goal of our study is to correlate the ratio of PR isoform expression, with the in vitro responsiveness of human breast cancer samples to MFP. Preliminary studies indicated that primary cultures of breast cancer samples overexpressing PR-A were responsive to MFP (10 nM) in vitro. However, since the rate of successful primary cultures was not encouraging (7%), we decided to develop a tissue culture assay to evaluate MFP responsiveness of breast cancer samples in vitro. Forty five breast cancer samples obtained from the General Pacheco´s Hospital, Buenos Aires, were kept in culture medium at room temperature and in dry ice. The former were cut in slices of 100 μm using a tissue chopper usually used to slice mouse nervous tissue for short incubations, and were incubated on filters in the presence of growth medium (fetal calf serum 10%) with or without MFP (10 nM), for 48 hs at 37º C. The slices were then fixed, paraffin embedded, and processed for Ki-67 staining to evaluate proliferating cells. Frozen samples were processed for Western blotting and the ratio of both PR isoforms was determined in nuclear extracts. In 17/45 (38%) of the samples valuable information was obtained. This was related with the quality (size and amount of viable cells) of the sample. The number of Ki-67 positive cells over the total viable cells was evaluated by a pathologist (M. May) in at least six different control or MFP-treated slices. From nine cases in which the Ki-67 index in control slices doubled the value obtained in MFP-treated slices (p<0.05), 8 were samples in which PR-A was higher than PR-B. MFP increased (p<0.05) the Ki-67 index in one sample with higher levels of PR-B than PR-A. No significant differences were observed in the remaining cases (null, equimolar or higher levels of PR-B than PR-A). We still plan to increase the number of samples in our study. Our data suggest that the tissue culture method developed is much more efficient than the primary cultures to evaluate drug or hormone responsiveness in breast cancer tissue. The results obtained herein support the proposal of PR-A as a predictive marker of antiprogestin therapy in breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-19.

Multidentate Zwitterionic Ligands Provide Compact and Highly Biocompatible Quantum Dots

Hydrophilic functional semiconductor nanocrystals that are also compact provide greatly promising platforms for use in bioinspired applications and are thus highly needed. To address this, we designed a set of metal coordinating ligands where we combined two lipoic acid groups, bis(LA)-ZW, (as a multicoordinating anchor) with a zwitterion group for water compatibility. We further combined this ligand design with a new photoligation strategy, which relies on optical means instead of chemical reduction of the lipoic acid, to promote the transfer of CdSe-ZnS QDs to buffer media. In particular, we found that the QDs photoligated with this zwitterion-terminated bis(lipoic) acid exhibit great colloidal stability over a wide range of pHs, to an excess of electrolytes, and in the presence of growth media and reducing agents, in addition to preserving their optical and spectroscopic properties. These QDs are also stable at nanomolar concentrations and under ambient conditions (room temperature and white light exposure), a very promising property for fluorescent labeling in biology. In addition, the compact ligands permitted metal-histidine self-assembly between QDs photoligated with bis(LA)-ZW and two different His-tagged proteins, maltose binding protein and fluorescent mCherry protein. The remarkable stability of QDs capped with these multicoordinating and compact ligands over a broad range of conditions and at very small concentrations, combined with the compatibility with metal-histidine conjugation, could be very useful for a variety of applications, ranging from protein tracking and ligand-receptor binding to intracellular sensing using energy transfer interactions.

Combining Ligand Design with Photoligation to Provide Compact, Colloidally Stable, and Easy to Conjugate Quantum Dots

We describe the design and synthesis of two compact multicoordinating (lipoic acid-appended) zwitterion ligands for the capping of luminescent quantum dots, QDs. This design is combined with a novel and easy to implement photoligation strategy to promote the in situ ligand exchange and transfer of the QDs to buffer media. This method involves the irradiation of the native hydrophobic nanocrystals in the presence of the ligands, which promotes in situ cap exchange and phase transfer of the QDs, eliminating the need for a chemical reduction of the dithiolane groups. Applied to the present LA-zwitterion ligands, this route has provided QDs with high photoluminescence yields and excellent colloidal stability over a broad range of conditions, including acidic and basic pH, in the presence of growth media and excess salt conditions. The small lateral extension of the capping layer allowed easy conjugation of the QDs to globular proteins expressing a terminal polyhistidine tag, where binding is promoted by metal-affinity interactions between the accessible Zn-rich surface and imidazoles in the terminal tag of the proteins. The ability to carry out conjugation in acidic as well as basic conditions opens up the possibility to use such self-assembled QD-protein conjugates in various biological applications.

EVALUATION OF ELECTROCHEMICAL CORROSION FOR MILD STEEL EXPOSED TO DIFFERENT CHLORIDE MEDIA.

The influence of different chloride media such as 3.5%NaCl, 10% NaCl, Vätäänen nine salt solutions (VNSS) prepared as artificial seawater and natural seawater on the corrosion of mild steel (MS) has been studied using electrochemical polarization techniques. The effect of the addition of starch, glucose, yeast extract, peptone, and mixture of growth media on the corrosion behavior of mild steel has been also discussed. The order of the rate of decrease in oxide film thinning proceeds according to the following order of the type of the applied medium: 10%NaCl > seawater > VNSS > 3.5%NaCl. On the other hand in case of mild steel immersed in VNSS in presence of different nutrients the rate of oxide film thinning proceed according to the following order of the type of growth medium: Peptone < glucose < yeast extract < mixture of growth media ≈ starch. Seawater was found to be less corrosive than VNSS. Addition of the growth media induced a decrease in both the cathodic and anodic currents and acted as a mixed type inhibitor. Cyclic polarization scans illustrate little decrease in the pitting tendency in the presence of growth media.

Atmospheric pressure-thermal desorption (AP-TD)/electrospray ionization-mass spectrometry for the rapid analysis of Bacillus spores

A technique is described where an atmospheric pressure-thermal desorption (AP-TD) device and electrospray ionization (ESI)-mass spectrometry (MS) are coupled and used for the rapid analysis of Bacillus subtilis spores in complex matrices. The resulting AP-TD/ESI-MS technique combines the generation of volatile compounds and/or pyrolysis products with soft-ionization MS detection. In the AP-TD/ESI-MS approach, an electrospray solvent plume was used as the ionization vehicle of thermally desorbed neutrals at atmospheric pressure prior to mass spectrometric analysis using a quadrupole ion trap mass spectrometer. The approach is quantitative with the volatile standard dimethyl methylphosphonate (DMMP) and with the use of an internal standard (diethyl methylphosphonate, DEMP). A linear response was obtained as tested in the 1-50 ppm range (R(2) = 0.991) with a standard error of the estimate of 0.193 (0.9% RSD, n = 5). Bacterial spores were detected by performing pyrolysis in situ methylation with the reagent tetramethylammonium hydroxide (TMAH) for the detection of the bacterial spore biomarker dipicolinic acid (DPA) as the dimethylated derivative (2Me-DPA). This approach allowed spore detection even in the presence of growth media in crude lyophilized samples. Repetitive analyses could be performed with a duty cycle of less than 5 min total analysis time (including sample loading, heating and data acquisition). This strategy proved successful over other direct ambient MS approaches like DESI-MS and AP-TD/ESI-MS without the in situ derivatization step to detect the dipicolinic acid biomarker from spores. A detection limit for the dimethylated DPA biomarker was estimated at 1 ppm (equivalent to 0.01 mug of DPA deposited in the thermal desorption tube), which corresponded to a calculated detection limit of 10(5) spores deposited or 0.1% by weight spore composition in solid samples (assuming a 1 mg sample size). The AP-TD/ESI source used in conjunction with the in situ methylation step allowed the differentiation of bacterial spores from other 'suspicious white powders' using a single stage for mass analysis and with minimum sample preparation, making this approach suitable for simple field-portable MS instrumentation and pattern recognition data analysis.

The fate of p21 CDKN1A in cells surviving UV-irradiation

p21 CDKN1A is a critical regulator of cell cycle progression in response to DNA damage. There are conflicting conclusions as to whether p21 CDKN1A levels increase or decrease after ultraviolet (UV)-irradiation and recently it was even reported to disappear entirely following 2.5–30 J m −2 of UV-irradiation in the presence of growth medium. The latter would suggest an alternative mechanism for cell cycle arrest after UV-irradiation, since p21 CDKN1A induction has been considered to be the major mediator of p53-mediated cell cycle arrest after DNA damage. Using physiological UV doses based on cell-killing, we previously observed and here verify that low doses (1.2–6 J m −2) induce p21 CDKN1A immediately after UV-irradiation, though higher doses cause a latency during which p21 CDKN1A levels remain fairly constant before increasing. As expected, p53 induction preceded p21 CDKN1A induction at all doses. Thus, p21 CDKN1A levels after low doses of UV-irradiation may be controlled in a p53-dependent manner without severe reduction. We propose that physiological relevant UV doses should be determined for each target cell type prior to studying UV-induced responses and that p21 CDKN1A is indeed critical for cell cycle arrest in cells that survive UV-irradiation.

Muscle differentiation going down the tube

Mammalian myotube terminal differentiation was previously believed to result in an irreversible differentiation process – evidenced by the downregulation of cyclins and cyclin-dependent kinases (CDKs) and through the expression of CDK inhibitors, retinoblastoma genes, muscle differentiation factors such as myogenin and contractile proteins such as troponin. However, Odelberg et al.1xDedifferentiation of mammalian myotubes induced by msx1. Odelberg, S.J et al. Cell. 2000; 103: 1099–1109Abstract | Full Text | Full Text PDF | PubMed | Scopus (325)See all References1 now report that a nuclear protein, msx1, can reverse myotube differentiation, and the resulting cells can be induced to differentiate into other cell types.The authors transduced the mouse msx1 gene into C2C12 myoblasts and found that the expression of msx1 prevented myotube formation. When msx1 was expressed in the presence of growth medium, the levels of MRF4, p21 and myogenin were reduced. This was followed by the reduction of MyoD expression. By plating myotubes at low density together with growth medium stimulation, they also showed that msx1 expression led to the cleavage of large myotubes into smaller tubes or proliferating mononucleated cells. The de-differentiated clones were then tested in different culture conditions for pluripotency. In chondrogenic differentiation medium, the de-differentiated myotube clones expressed collagen type II and could be further induced to express collagen type X. In adipogenic differentiation medium, adipocyte morphology was induced and numerous vacuoles stained positive in the presence of lipophilic dyes. In osteogenic medium, in addition to foci staining with alkaline phosphatase, osteogenic markers such as osteocalcin and osteopontin were detected. Furthermore, when msx1expression was deliberately switched off, chondrogenic, adipogenic, myogenic or osteogenic markers could still be detected under various differentiation conditions, suggesting that trans-determination had occurred.These data show that mammalian myotubes can be de-differentiated, and the de-differentiated cells have properties characteristic of stem cells. As the authors point out, the molecular mechanisms underlying msx1 activities are still unknown. It remains to be discovered whether msx1 can interfere with factors that affect maintenance of differentiation and in so doing regulate the inheritance of different differentiation states.

Proliferation and extracellular matrix production by human infragenicular smooth muscle cells in response to interleukin-1β

Purpose: Atherosclerotic peripheral vascular disease commonly involves the infragenicular arterial tree. Our study evaluated the effect of interleukin (IL)-1β on the proliferation of vascular smooth muscle cells (VSMCs) derived from atherosclerotic infragenicular arteries of human subjects who underwent below-knee amputation, as well as the role of IL-1β in VSMCs' production of extracellular matrix components, substances that are important in the transformation of VSMCs from the contractile to the synthetic phenotype. This transformation to the synthetic phenotype is an important step in the formation of the atherosclerotic lesion. Methods: Cultures were identified as being of smooth muscle origin through staining with the cytoskeletal marker, α–smooth muscle actin. Proliferation assays were performed by seeding confluent cultures of passages 4 to 7 into six-well plates at 10,000 cells per well. After serum starvation, samples were incubated with IL-1β (1 ng/ml). Cell number was determined on a daily basis. To study extracellular matrix production, cells were propagated in tissue culture chamber slides in the absence or presence of growth media containing IL-1β. After fixation with 100% methanol, each sample was stained with a primary antibody specific for an extracellular matrix component. After staining with the fluorescein-tagged secondary antibody, each sample was examined using immunofluorescent microscopic examination. Results: The results of our proliferation assays showed that IL-1β caused a significant increase in the proliferation of VSMCs at 24, 48, 72, and 96 hours (p H 0.003 when comparing IL-1β–treated samples with control specimens at each time period using unpaired t test). The number of IL-1β–treated cells at 96 hours was double the number present in the control samples (16,033 ± 238 vs 8102 ± 824). When compared with control samples, IL-1β was found to affect the production of extracellular matrix proteins by infragenicular VSMCs. IL-1β caused an increase in the production of fibronectin, a decrease in the production of laminin, and no change in the production of collagen type IV. Conclusions: These results suggest that interleukin-1β acts as a potent stimulant of the proliferation of human infragenicular VSMCs. IL-1β also acts to augment the production of fibronectin by these cells. Fibronectin has been implicated in the phenotypic transformation of VSMCs from the contractile to the synthetic state. Therefore, IL-1β may serve as an important regulatory factor in the development of atherosclerosis by stimulating the proliferation of VSMCs and their transformation to the synthetic state, two important steps in the formation of the atherosclerotic lesion. (J Vasc Surg 1997;26:1002-8.)

Cytosolic 85-kDa Phospholipase A2-mediated Release of Arachidonic Acid Is Critical for Proliferation of Vascular Smooth Muscle Cells

Recent evidence suggests that arachidonic acid (AA) may be involved in regulating cellular proliferation. The predominant mechanism of AA release from cellular phospholipids is via phospholipase A2 (PLA2) hydrolysis. The purpose of this study was to examine the roles of the distinct 14-kDa and 85-kDa PLA2 enzymes in human coronary artery vascular smooth muscle cell (hCAVSMC) proliferation. Cultured hCAVSMCs proliferate in the presence of growth medium with a typical doubling time of 30-40 h, grow at a slower proliferative rate upon reaching confluency (day 8), and eventually undergo contact inhibition of growth (day 10). Neither Type II 14-kDa PLA2 activity nor mass changed over a 10-day culture period. In contrast, 85-kDa PLA2 protein activity and mRNA decreased as time in culture progressed. This reduction in 85-kDa PLA2 correlated with reductions in DNA synthesis and suggested a possible association between 85-kDa PLA2 and proliferation. To directly evaluate the role of the 85-kDa PLA2 in proliferation we examined the effects of an 85-kDa PLA2 inhibitor (AACOCF3) and 85-kDa PLA2 antisense oligonucleotides on proliferation. Both reagents dose dependently inhibited proliferation, whereas a 14-kDa PLA2 inhibitor (SB203347), a calcium-independent PLA2 inhibitor (HELSS), an 85-kDa sense oligonucleotide, and a nonrelevant scrambled control oligonucleotide had no effect. The mechanism by which 85-kDa PLA2 influences cellular proliferation remains unclear. Inhibition of 85-kDa PLA2 activity produced neither phase-specific cell cycle arrest nor apoptosis (fluorescence-activated cell sorter analysis). Addition of AA (20 mu M) attenuated the effects of both AACOCF3 and 85-kDa antisense oligonucleotides implicating AA as a key mediator in cellular proliferation. However, although prostaglandin E2 (PGE2) was present in the culture medium, it peaked early (day 3) in culture, and indomethacin had no effect on cellular proliferation indicating that hCAVSMC proliferation was not mediated through PGE2. These data provide the first direct evidence that PLA2 is involved in control of VSMC proliferation and indicate that 85-kDa PLA2-mediated liberation of AA is critical for cellular proliferation.

Biosynthesis of interleukin-6 by cultured human chorion laeve cells: regulation by cytokines.

Intrauterine infection is an important cause of preterm labor and delivery and is characterized by increased production of inflammatory cytokines by gestational tissues. We evaluated the biosynthesis of the inflammatory cytokine interleukin-6 (IL-6) by human chorion laeve cells and its regulation by other cytokines essential to the inflammatory process. We found that cultured chorion cells secrete IL-6 in the presence of growth medium supplemented only with 10% fetal calf serum. IL-1 beta, tumor necrosis factor, and lipopolysaccharide all induced a significant concentration-dependent stimulation of IL-6 production by chorion cells. The concentration range of each cytokine tested (0.1-10 ng/mL) is within the range of values found in the amniotic fluid of women destined to deliver preterm due to infection of gestational tissues. Additionally, treatment of chorion cells with IL-1 beta in combination with actinomycin-D or cycloheximide attenuated the stimulatory action of IL-1 beta on IL-6 production. Northern blot analysis of total RNA from cultured chorion cells stimulated with IL-1 beta demonstrated that IL-6 mRNA increases over time. Our data suggest that IL-6 is produced by human fetal chorion in response to infection of maternal gestational tissues. In conjunction with other inflammatory mediators, fetally derived IL-6 may play a role in the pathophysiology of preterm labor due to infection.

HCO 3 − Fixation by Naturally Occurring Tufts and Pure Cultures of Thiothrix nivea

Naturally occurring tufts of the mixotroph Thiothrix nivea blanketed the East Everglades (Dade County, Fla.) Chekika artesian well and runoff areas. The rate of HCO(3) fixation by these Thiothrix tufts was determined to be 14.0 +/- 5.4 nmol of HCO(3) per min per mg of dry weight, which reflected a growth rate of 5.0%/h. The addition of 10 mM glucose, ribose, acetate, or pyruvate or 0.05% Casamino Acids (Difco Laboratories, Detroit, Mich.) did not appear to alter the HCO(3) fixation rate. Whereas 1 mM acetate or 10 mM lactate, ethanol, glycerol, alpha-ketoglutarate, succinate, fumarate, or citrate slightly stimulated HCO(3) fixation, 5 to 10 mM malate inhibited HCO(3) fixation by 90%. Pure Thiothrix cultures isolated from Chekika fixed HCO(3) at rates as high as 29.9 +/- 2.8 nmol of HCO(3) per min per mg of dry weight in the presence of growth medium. Malate did not have a suppressive effect but rather slightly stimulated in vivo HCO(3) fixation.

Expression of MyoD1 coincides with terminal differentiation in determined but inducible muscle cells.

We have examined the expression of MyoD1, a potential determination factor of myogenic cells, in permissive and inducible C2 myoblasts. These two types of myoblasts exhibit distinct requirements to undergo terminal differentiation. Unlike permissive cells, inducible cells fail to differentiate in the presence of growth medium plus fetal calf serum and require insulin to undergo terminal differentiation. We show that while expression of MyoD1 is constitutive in permissive cells, no trace of MyoD1 transcripts is found in inducible cells at the myoblast stage. In these cells, however, expression of MyoD1 accompanies differentiation. This indicates that MyoD1 may not be required for the maintenance of the myoblast phenotype, and could act as an effector of terminal differentiation in already determined muscle cells. Our results provide new evidence that permissive and inducible cells represent two distinct stages of the progression of determined muscle cells toward terminal differentiation.

Sources of low-molecular-weight host factors that phenotypically increase the resistance of Haemophilus influenzae type b to bacteriolysis: non-identity with a factor active in gonococci

Both gonococci and Haemophilus influenzae type b (Hib) in response to incubation with low-molecular-weight components of blood are converted to a relative resistance to killing by antibody and complement in vitro. Recently, cytidine 5′-mono-phospho-N-acetylneuraminic acid (a sialyl donor present in mammalian tissues) was found to convert gonococci. Here we report that this compound was inactive in Hib. In further contrast, the Hib-converting activity of serum was not retained by 500-M r filters, not sensitive to pH 2, and not active in the presence of growth media. Thus the phenotypic resistances in Hib and gonococci appear to have a different metabolic basis. The low-M r serum factors active in Hib remain to be identified. Filtrates of human nasopharyngeal washes were also active, further suggesting that the converted form resembles the phenotype of Hib in vivo.

Involvement of multiple subcellular compartments in intracellular processing of epidermal growth factor.

The intracellular translocation and processing of epidermal growth factor (EGF) in 3T3 cells has been studied utilizing Percoll density gradients. EGF is internalized and rapidly becomes associated with two types of intracellular compartments. The extent to which EGF is delivered to these two compartments is apparently regulated depending upon the cell's physiological condition. In growth medium, an increased proportion of EGF is taken up into a Golgi-like element. Uptake through this pathway correlates with a decrease in degradation of the ligand. In the absence of serum and amino acids, an increased proportion of EGF is taken up into a component which has a density of 1.05. Uptake through this pathway correlates with increased degradation of the ligand. The ligand taken up through both pathways is transferred to dense vesicles which comigrate with lysosomes. In the presence of growth medium, however, dense vesicles containing EGF can be shown to be lysosomal enzyme-deficient upon further fractionation. In addition, in the presence of serum, a portion of the internalized EGF is apparently released from the cells, intact, and then re-bound. The processes described may be important in the production of a mitogenic response and the ability of cells to self-regulate their responsiveness to the growth factor.

Effects of Dimethyl Sulfoxide on Tetrahymena pyriformis GL. Fine Structural Changes and Their Reversibility*

Fine-structural changes are induced in Tetrahymena by exposure to 7.5% dimethyl sulfoxide (DMSO) in the presence of growth medium. Some of these changes (nucleolar, mitochondrial, peroxisomal) resemble those seen during starvation, in agreement with the previously reported inhibitory effect of DMSO on food-vacuole formation; however, changes such as helical formations of polyribosomes indicate additional internal actions of the reagent. The effects vary to some extent within the same group of cells, suggesting that sensitivity to the reagent may differ with the stage in the cell cycle. The structural changes induced by a 1-hr exposure to DMSO are reversible, but recovery of the cells after removal of the reagent is slower than that seen after starvation. The observations suggest that the recovery is associated with renewed synthesis.

Regulation of glycoconjugate expression on cloned mammalian cells: Studies on the growth dependence of membrane-associated/blood group H antigen

Studies of HeLa S-3 cell clones derived from suspensions of single cells were carried out to determine the relationship of clonal growth patterns to blood group H antigen expression. Cloning efficiency of these cells was excellent in the presence of growth medium which contained pooled human serum. At the end of the growth period of 9 to 11 days, three quarters of the clones were medium sized (cell counts ranged from 100–500), or large (greater than 500 cells), and approximately one fourth were classified as small (less than 100 cells). A mixture of H positive and H negative cells was observed in medium and large clones; therefore the composition of such clones resembled the cellular blood group composition of the original suspension (50% H positive, 50% H negative). Small clones contained all H-negative cells or only a few H-positive cells. Optimal expression of H antigen thus appeared to depend upon a growth pattern similar to that observed in exponentially growing monolayer cells. Clones at the 2 to 8 cell stage were mostly H-negative; this finding was consistent with the view that alterations in culture technique may have caused transient cellular and enzyme alterations which in the case of H antigen was reflected by a temporary loss of phenotype.