Proteomic markers of low and high fertility bovine spermatozoa separated by Percoll gradient.

In the context of artificial insemination, male fertility is defined as the ability to produce functional spermatozoa able to withstand cryopreservation. We hypothesized that interindividual variations in fertility depend on the proportion of the fully functional sperm population contained in the insemination dose. The objective of this study was to identify protein markers of the fully functional sperm subpopulation. Insemination doses from four high-fertility (HF) and four low-fertility (LF) bulls with comparable post-thaw quality parameters were selected for proteomic analysis using iTRAQ technology. Thawed semen was centrifuged through a Percoll gradient to segregate the motile (high density [HD]) from the immotile (low density [LD]) sperm populations. Sperm proteins were extracted with sodium deoxycholate and four groups were compared: LD and HD spermatozoa from LF and HF bulls. A total of 498 unique proteins were identified and quantified. Comparison of HD spermatozoa from HF and LF bulls revealed that five proteins were significantly more abundant in the HF group (AK8, TPI1, TSPAN8, OAT, and DBIL5) whereas five proteins were more abundant in the LF group (RGS22, ATP5J, CLU, LOC616319, and CCT5). Comparison of LD spermatozoa from HF and LF bulls revealed that four proteins were significantly more abundant in the HF group (IL4I1, CYLC2, OAT, and ARMC3) whereas 15 proteins were significantly more abundant in the LF group (HADHA, HSP90AA1, DNASE1L3, SLC25A20, GPX5, TCP1, HIP1, CLU, G5E622, LOC616319, HSPA2, NUP155, DPY19L2, SPERT, and SERPINE2). DBIL5, TSPAN8, and TPI1 showed potential as putative markers of the fully functional sperm subpopulation.

Reduction of Trypanosoma equiperdum from equine semen by single layer centrifugation.

Trypanosoma equiperdum (T. equiperdum) causes dourine, a venereally transmitted infection in horses. Purification of semen by single layer centrifugation (SLC) has been proven to be successful in reducing venereally transmitted diseases when dealing with other pathogens. The objective of this study was to evaluate the purification of T. equiperdum spiked semen by SLC. Semen was spiked using cryopreserved T. equiperdum stabilates (Dodola strain isolate 943). In total, 6 concentrations, varying from 102 to >5 × 106 trypanosomes, were added to semen samples. Subsequently, SLC was performed following standard procedures. The presence of the parasite in the purified semen was checked by wet smear examination, ITS1 PCR and in vivo inoculation in mice. Before SLC, all spiked semen samples, except the negative controls, were positive on PCR analysis. After SLC, all the pellets were found to be negative for T. equiperdum on microscopic examinations. Examination of the pellet by PCR could also not detect any parasite-DNA in the SLC-pellet of semen spiked with the lower number of parasites (102 to104 trypanosomes). However, in the SLC pellets spiked with 104 - 5 × 104 trypanosomes, only 1 out of the 4 replicates was negative for parasite DNA. All groups spiked with >5 × 104 trypanosomes were found to be positive on PCR. All mice in the positive controls exhibited parasitaemia (5/5). Mice inoculated with SLC-purified semen that was spiked with lower than 5 × 104 trypanosomes, remained free of parasitaemia, similar to the negative controls. However inoculation with SLC-pellets from samples with a higher number of trypanosomes (>5 × 104 - 5 × 106 and > 5 × 106), induced parasitaemia in 2 out of 5 and 3 out of 5 mice, respectively. This study indicates that single layer centrifugation can be used to clear T. equiperdum infected semen but that the success is dependent on the number of parasites.

Effect of rotor type on the separation of isotope-labeled and unlabeled Escherichia coli RNA by isopycnic density ultracentrifugation.

Separation of differentially isotope-labeled bacterial RNA by isopycnic density gradient centrifugation is a critical step in RNA-based stable isotope probing analyses, which help to link the structure and function of complex microbial communities. Using isotope-labeled Escherichia coli RNA, we showed that an 8 mL near-vertical rotor performed better than a 2 mL fixed-angle rotor, thereby corroborating current recommendations. Neither increased concentrations of formamide nor urea in the medium improved the separation results using the fixed-angle rotor.

Impact of the virus purification protocol on aggregation and electrokinetics of MS2 phages and corresponding virus-like particles.

Previous experimental and theoretical studies have established that electrokinetic and aggregation properties of soft MS2 phages are not only governed by the physico-chemical features of their proteinaceous outer surface but are also significantly impacted by those of their inner RNA component (Dika et al. Appl. Environ. Microbiol., 2011, 14, 4939-4948). These conclusions contradict the recent findings of Nguyen et al. (Soft Matter, 2011, 7, 10449-10456) who reported identical electrokinetic and aggregation characteristics for MS2 and corresponding virus like particles (VLPs) that lack the internal RNA component. We demonstrate here that this contradiction originates from the different purification methods adopted prior to measurements. More generally, we show that stability and electrohydrodynamics of viruses differ according to purification by (i) dialysis, (ii) isopycnic centrifugation in the cesium chloride gradient, and (iii) precipitation using polyethylene glycol (PEG). Methods (i) and (iii) lead to aggregation of MS2 phages at pH ≤ 4 and pH ≤ 6 in 1-100 mM NaNO3 solutions, respectively, while under such conditions aggregation is not observed for MS2 and VLP suspensions prepared according to (ii). In addition, VLPs prepared following methods (i) and (iii) aggregate only at the isoelectric point (pH ~ 3-4) in 1 mM NaNO3 solution. Electrophoretic mobility data of stable MS2 and VLP particles were further examined using a recent formalism for electrokinetics of soft multilayered colloids. The analysis qualitatively shows how the purification protocol may affect either the outer particle surface properties and/or the inner particle content. Finally, the non-DLVO aggregation behavior of MS2 and VLPs purified via the above protocols is discussed in terms of the possible change in corresponding interparticular interactions.

Identification of cellulose-responsive bacterial and fungal communities in geographically and edaphically different soils by using stable isotope probing.

Many bacteria and fungi are known to degrade cellulose in culture, but their combined response to cellulose in different soils is unknown. Replicate soil microcosms amended with [(13)C]cellulose were used to identify bacterial and fungal communities responsive to cellulose in five geographically and edaphically different soils. The diversity and composition of the cellulose-responsive communities were assessed by DNA-stable isotope probing combined with Sanger sequencing of small-subunit and large-subunit rRNA genes for the bacterial and fungal communities, respectively. In each soil, the (13)C-enriched, cellulose-responsive communities were of distinct composition compared to the original soil community or (12)C-nonenriched communities. The composition of cellulose-responsive taxa, as identified by sequence operational taxonomic unit (OTU) similarity, differed in each soil. When OTUs were grouped at the bacterial order level, we found that members of the Burkholderiales, Caulobacteriales, Rhizobiales, Sphingobacteriales, Xanthomonadales, and the subdivision 1 Acidobacteria were prevalent in the (13)C-enriched DNA in at least three of the soils. The cellulose-responsive fungi were identified as members of the Trichocladium, Chaetomium, Dactylaria, and Arthrobotrys genera, along with two novel Ascomycota clusters, unique to one soil. Although similarities were identified in higher-level taxa among some soils, the composition of cellulose-responsive bacteria and fungi was generally unique to a certain soil type, suggesting a strong potential influence of multiple edaphic factors in shaping the community.

Do it yourself! - Initial experiences with self-synthesized CsTFA for RNA-SIP analyses.

Cesium trifluoroacetate (CsTFA) is a gradient medium for isopycnic centrifugation in RNA-based Stable Isotope Probing (RNA-SIP), an important means to link the structure and function of microbial communities. We report a protocol to easily synthesize CsTFA from cesium carbonate (Cs2CO3) and trifluoroacetic acid (TFA) and show that self-synthesized CsTFA performs similarly to commercial CsTFA in the separation of isotopically labelled and unlabelled bacterial RNA.

Expression of E1E2 on hepatitis C RNA-containing particles released from primary cultured human hepatocytes derived from infected cirrhotic livers.

OBJECTIVE: To determine whether liver-derived hepatitis C RNA-containing particles express the E1E2 discontinuous antigenic determinant defined by unique monoclonal antibody (mAb) D32.10 which recognizes three highly conserved segments in E1 (aa297-306) and E2 (aa480-494 and aa613-621) envelope glycoproteins. METHODS: Human hepatocytes were isolated from HCV-infected cirrhotic explanted livers. The liver-derived hepatitis C virus (HCV) particles released from three distinct cultures (genotypes 1b and 2b) were characterized. HCV RNA+ was quantified by real-time RT-PCR. The E1E2 antigenic activity was assessed by indirect ELISA and immunoblotting using D32.10. The density distributions of HCV RNA and E1E2 antigen were determined by isopycnic sucrose density gradients. HCV E1E2, E2 and core antigens were detected in the cells by immunochemical staining. RESULTS: Liver-derived HCV particles contained HCV RNA (106-107 copies/mg of protein) and core proteins and expressed the E1E2/D32.10 epitope. HCV RNA and E1E2 cosedimented between 1.15 and 1.25 g/ml in sucrose gradients. Moreover, the mAb D32.10 detected E1E2 by immunostaining in HCV-infected hepatocytes in parallel with E2 and core antigens. CONCLUSION: Our results provide evidence that the mAb D32.10 recognizes E1E2 envelope complexes expressed in the cell cytoplasm and on the surface of HCV RNA-containing particles released from short-term cultures of in vivo infected hepatocytes.

Isolation and Quality Control of Functional Mitochondria.

Even in times, when the study of mitochondria in their natural cellular context is becoming more and more popular, some scientific questions still require the preparation of isolated mitochondria. Numerous protocols are available being adapted for different cell or tissue types allowing isolation of "pure" mitochondria trying to preserve their "structural and functional" integrity. In this chapter, we intend to provide a more general framework introducing differential isopycnic density gradient centrifugation strategy with a special focus sensitizing for the specific challenges coming along with this method and how to obtain "functional," enriched, "intact" mitochondria. Due to the fact that in any study dealing with these organelles standardized processing is mandatory, here we describe a strategy addressing quality control of prepared intact mitochondria. The quality control should be an integrated part of all isolation processes. The underlying protocol should be seen as starting point and has to be carefully adjusted to cover different sample types used for the diverse research questions.

Dissociation between mitogenicity and immunogenicity of TNP-lipopolysaccharide, a T-independent antigen.

Two models have been proposed to explain triggering of B cells by so-called "T-independent antigen." Feldmann and Basten (1) proposed that the interaction of multiple repeating determinants on polymeric antigens with specific Ig receptors on the B-cell surface is sufficient to provide the signals for division of these cells and differentiation to antibody-forming cells. In contrast, coutinho et al. (2, and see review, 3) have claimed that there is only one signal, a mitogenic signal, receptors acting merely as passive focusing devices to localize the antigen on specific cells where it delivers a mitogenic signal resulting in differentiation to an antibody-producing cell. This model rests primarily on the demonstration that at high concentration all T-independent antigens they have tested are mitogenic for B cells (4-6). Compatible with this hypothesis are the observations that hydrolysis of lipopolysaccharide (LPS) to remove the ester- linked fatty acids of the mitogenic lipid A component abrogates its mitogenic (7,8) activity as well as its ability, when substituted with the TNP hapten, to induce a T-independent anti- TNP response (9). However, alkali treatment of LPS, although not changing its antigenic component (8), may also modify the molecule physically or chemically which could account for loss of immunogenic properties (10). We therefore investigated other reagents which interact with LPS in a more chemically defined manner in an effort to clarify the relationship between the mitogenic and immunogenic properties of this molecule. Polymyxin B (PB) is one of a family of cyclic peptide antibiotics which are bactericidal for most gram-negative bacteria. It prevents the lethal endotoxic activity of LPS (11, 12) and changes the physical structure of LPS (13). We report here that low doses of PB added to cultures of mouse spleen cells inhibit the mitogenic activity of TNP-LPS, a T- independent antigen, and native LPS, but do not suppress the immune response to TNP-LPS. PB interacts with TNP-LPS and LPS causing a physical change in the molecule. In addition, polymyxin-treated LPS is no longer mitogenic. These results suggest a dissociation between the mitogenic and immunogenic properties of TNP-LPS.

Analytical subcellular fractionation of cultivated mouse resident peritoneal macrophages.

Resident peritoneal macrophages of the mouse, cultivated for 3 d, have been studied by quantitative subcellular fractionation using differential centrifugation and density equilibration in linear gradients of sucrose. Density equilibration experiments were carried out on untreated cytoplasmic extracts, on cytoplasmic extracts treated with digitonin or sodium pyrophosphate, and on cytoplasmic extracts derived from cells cultivated for 24 h in the presence of Triton WR-1339. The enzyme distributions obtained distinguished six typical behaviors characteristic of distinct subcellular entities. Acid alpha-galactosidase and other acid hydrolases displayed the highest average velocity of sedimentation and equilibrium density. Culturing in a medium that contained Triton WR-1339 markedly decreased their density, most likely as a result of Triton WR-1339 accumulation within lysosomes. Cytochrome c oxidase and the sedimentable activity of malate dehydrogenase showed a narrow density distribution centered around 1.17, very similar under all the experimental situations; their rate of sedimentation fell within the range expected for mitochondria. Catalase was particle-bound and exhibited structure-linked latency (80 percent); it was released in soluble and fully active form by digitonin, but this required a much higher concentration than in the case of lysosomal enzymes. Differences relative to all the other enzymes studied suggest the existence of a particular species of organelles, distinctly smaller than mitochondria, and possibly related to peroxisomes. Many enzymes were microsomal in the sense that the specific activities, but not the yields, were greater in microsomes than in other fractions obtained by differential centrifugation. These enzymes were distinguished in three groups by their properties in density equilibration experiments. NAD glycohydrolase, alkaline phosphodiesterase I, and 5'-nucleotidase had low equilibrium densities but became noticeably more dense after addition of digitonin. The other microsomal enzymes were not shifted by digitonin, in particular N-acetylglucosaminyltransferase and galactosyltransferase, which otherwise equilibrated at the same position in the gradient. We assign the digitonin-sensitive enzymes to plasma membranes and possibly to related endomembranes of the cells, and the two glycosyltransferases to elements derived from the Golgi apparatus. Finally, alpha-glucosidase, sulphatase C, NADH cytochrome c reductase, NADPH cytochrome c reductase, and mannosyltransferase, equilibrated at a relatively high density but were shifted to lower density values after addition of sodium pyrophosphate. These properties support their association with elements derived from the endoplasmic reticulum.

Subcellular localization and heterogeneity of neutral proteases in neutrophilic polymorphonuclear leukocytes.

The subcellular localization of elastase and of neutral proteases hydrolyzing histone and casein was determined in human and rabbit polymorphonuclear leukocytes using fractionation by isopycnic centrifugation. Granule-rich fractions obtained by this technique were extracted and analyzed by acrylamide gel electrophoresis, and proteolytic activity on the gels was demonstrated by staining with either N-acetyl-D,L-alanine alpha-naphthyl ester or naphthol AS-D acetate as substrate. In both species, all neutral proteases assayed were found to be localized exclusively in the azurophil granules. Specific activities were about 10-30 times higher in human than in rabbit preparations. In extracts of human azurophil granules up to 10 proteins exhibiting esterolytic activity could be demonstrated after electrophoretic separation. Three major and two or three minor components of these esterases were shown to possess elastase activity. Similar zymograms prepared with extracts from rabbit azurophil granules revealed only one major elastase band. The electrophoretic analysis further showed that the most strongly cationic proteins of both human and rabbit PMNs were also confined to the azurophil granules.

Proteomic analysis of defined HDL subpopulations reveals particle-specific protein clusters: relevance to antioxidative function.

OBJECTIVE: Recent proteomic studies have identified multiple proteins that coisolate with human HDL. We hypothesized that distinct clusters of protein components may distinguish between physicochemically-defined subpopulations of HDL particles, and that such clusters may exert specific biological function(s). METHODS AND RESULTS: We investigated the distribution of proteins across 5 physicochemically-defined particle subpopulations of normolipidemic human HDL (HDL2b, 2a, 3a, 3b, 3c) fractionated by isopycnic density gradient ultracentrifugation. Liquid chromatography/electrospray mass spectrometry identified a total of 28 distinct HDL-associated proteins. Using an abundance pattern analysis of peptide counts across the HDL subfractions, these proteins could be grouped into 5 distinct classes. A more in-depth correlational network analysis suggested the existence of distinct protein clusters, particularly in the dense HDL3 particles. Levels of specific HDL proteins, primarily apoL-I, PON1, and PON3, correlated with the potent capacity of HDL3 to protect LDL from oxidation. CONCLUSIONS: These findings suggest that HDL is composed of distinct particles containing unique (apolipo)protein complements. Such subspeciation forms a potential basis for understanding the numerous observed functions of HDL. Further work using additional separation techniques will be required to define these species in more detail.

Transgenic, fluorescent Leishmania mexicana allow direct analysis of the proteome of intracellular amastigotes.

Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material. This has also precluded direct proteome analysis of the intracellular, amastigote form of Leishmania spp., protozoan parasites that cause a spectrum of diseases that affect some 12 million patients worldwide. Here a method is presented that combines classic, isopycnic density centrifugation with fluorescent particle sorting for purification by exploiting transgenic, fluorescent parasites to allow direct proteome analysis of the purified organisms. By this approach the proteome of intracellular Leishmania mexicana amastigotes was compared with that of extracellular promastigotes that are transmitted by insect vectors. In total, 509 different proteins were identified by mass spectrometry and database search. This number corresponds to approximately 6% of gene products predicted from the reference genome of Leishmania major. Intracellular amastigotes synthesized significantly more proteins with basic pI and showed a greater abundance of enzymes of fatty acid catabolism, which may reflect their living in acidic habitats and metabolic adaptation to nutrient availability, respectively. Bioinformatics analyses of the genes corresponding to the protein data sets produced clear evidence for skewed codon usage and translational bias in these organisms. Moreover analysis of the subset of genes whose products were more abundant in amastigotes revealed characteristic sequence motifs in 3'-untranslated regions that have been linked to translational control elements. This suggests that proteome data sets may be used to identify regulatory elements in mRNAs. Last but not least, at 6% coverage the proteome identified all vaccine antigens tested to date. Thus, the present data set provides a valuable resource for selection of candidate vaccine antigens.

Isolation of Glycosomes from Trypanosoma cruzi.

Glycosomes are peroxisome-related organelles of trypanosomatids in which the glycolytic and some other metabolic pathways are compartmentalized. We describe here two methods for the purification of glycosomes from Trypanosoma cruzi for preparative purposes, differential and isopycnic centrifugation. These are two techniques that allow the separation of different cellular compartments based on their different physicochemical characteristics. The first type of centrifugation is a rapid method that does not require large inputs and allows for fractions enriched in specific cell compartments to be obtained. The second type of centrifugation is a more elaborate method, but enables highly purified cellular compartments to be isolated. The success in obtaining these purified, intact organelles critically depends on using an appropriate method for controlled rupture of the cells.

Subcellular compartmentalization of maize storage proteins in Xenopus oocytes injected with zein messenger RNAs.

Maize storage proteins synthesized in oocytes were compartmentalized in membrane vesicles because they were resistant to hydrolysis by protease, unless detergent was present. The site of storage protein deposition within the oocyte was determined by subcellular fractionation. Optimal separation of oocyte membranes and organelles was obtained when EDTA and high concentrations of NaCl were included in the homogenization and gradient buffers. Under these conditions, fractions in sucrose gradients containing a heterogeneous mixture of smooth membranes (presumably endoplasmic reticulum, Golgi apparatus, and plasma membrane, density = 1.10-1.12 g/cm3), mitochondria (densities = 1.14 and 1.16 g/cm3), yolk platelets (density = 1.21 g/cm3), and a dense matrix material (density = 1.22 g/cm3) could be separated. Some zein proteins were recovered in the mixed membrane fraction, but the majority occurred in vesicles sedimenting with yolk platelets and granular material at a density of approximately 1.22 g/cm3. When metrizamide was included in the gradient to increase the density, little of the dense matrix material was isolated, and vesicles containing zein proteins were separated from other oocyte components. These vesicles were similar to protein bodies in maize endosperm because they were of identical density and contained the same group of polypeptides.

Mitochondrial ribosome assembly in Neurospora. Structural analysis of mature and partially assembled ribosomal subunits by equilibrium centrifugation in CsCl gradients.

In Neurospora, one protein associated with the mitochondrial small ribosomal subunit (S-5, Mr 52,000) is synthesized intramitochondrially and is assumed to be encoded by mtDNA. When mitochondrial protein synthesis is inhibited, either by chloramphenicol or by mutation, cells accumulate incomplete mitochondrial small subunits (CAP-30S and INC-30S particles) that are deficient in S-5 and several other proteins. To gain additional insight into the role of S-5 in mitochondrial ribosome assembly, the structures of Neurospora mitochondrial ribosomal subunits, CAP-30S particles, and INC-30S particles were analyzed by equilibrium centrifugation in CsCl gradients containing different concentrations of Mg+2. The results show (a) that S-5 is tightly associated with small ribosomal subunits, as judged by the fact that it is among the last proteins to be dissociated in CsCl gradients as the Mg+2 concentration is decreased, and (b) that CAP-30S and INC-30S particles, which are deficient in S-5, contain at most 12 proteins that are bound as tightly as in mature small subunits. The CAP-30S particles isolated from sucrose gradients contain a number of proteins that appear to be loosely bound, as judged by dissociation of these proteins in CsCl gradients under conditions in which they remain associated with mature small subunits. The results suggest that S-5 is required for the stable binding of a subset of small subunit ribosomal proteins.

Transient N-acetylglucosamine in the biosynthesis of phytohemagglutinin: attachment in the Golgi apparatus and removal in protein bodies.

Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the lectin phytohemagglutinin (PHA) during seed development. The polypeptides of PHA are synthesized by endoplasmic reticulum-bound polysomes and co-translationally glycosylated, pass through the Golgi complex, and accumulate in protein bodies, which constitute the lysosomal compartment in these cells. Some of the high-mannose sidechains of PHA are modified in the Golgi complex, and in mature PHA they contain N-acetylglucosamine, mannose, fucose, and xylose in the molar ratios 2, 3.8, 0.6, and 0.5. The results reported here show that the Golgi complex is also the site of additional N-acetylglucosamine incorporation into the modified sidechains. When developing cotyledons are labeled with [3H]glucosamine and glycopeptides of PHA present in the Golgi complex isolated, the radioactivity can be released as [3H]N-acetylglucosamine by digestion of the glycopeptides with beta-N-acetylglucosaminidase, indicating that the residues are in a terminal position. Arrival of PHA in the protein bodies is followed by the slow removal of these terminal N-acetylglucosamine residues, resulting in a decrease in the Mr of the modified sidechains. The biosynthetic intermediates of the glycoproteins destined for the lysosomal compartments of animal cells contain high-mannose sidechains modified by phosphate groups covered by N-acetylglucosamine that is labile to mild acid treatment. When cotyledons are labeled with [32P]orthophosphate, there is no radioactivity in PHA obtained from any of the subcellular fractions. There is also no release of radioactivity when [3H]glucosamine-labeled glycopeptides obtained from PHA in the Golgi complex are subjected to mild acid hydrolysis. These results indicate that the sorting-signals and posttranslational processing steps for proteins that are transported to the lysosomal compartment are different in plant cells and animal cells.

Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures.

Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies. Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes. One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point. This component was conspicuously labeled by [3H]proline in vivo. The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan. Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin. Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin. We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin. Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane. This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion. The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues. However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated. This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.

Arginine deprivation in KB cells. II. Characterization of the DNA synthesized during starvation.

DNA synthesis in cells deprived of arginine was examined. Three lines of evidence indicated that tritiated thymidine ([3H]TdR) incorporation in arginine-starved cells was due to replicative rather than repair DNA synthesis. (a) When made in the presence of bromodeoxyuridine, the [3H]TdR-labeled DNA sedimented at hybrid density in isopycnic gradients. (b) As determined by the diphenylamine reaction, there was a 15% increase in the chemical amount of DNA per culture 30 h after arginine deprivation. (c) [3H]TdR incorporation was hydroxyurea-sensitive. Alkaline velocity sedimentation of the total DNA made during starvation revealed the existence of two distinct size classes: most of the DNA sedimented at a position analogous to that of control DNA, but 40% migrated one-third the distance of the bulk. After arginine restoration, these shorter pieces appeared to be chased into DNA of normal length; thus, one lesion in deprived cultures may cause an arrest in completion of DNA stretches to mature size. These findings, together with results of morphological studies of starved cells, suggest that changes induced by arginine deficiency effect the organization of nucleoproteins. These changes are reversible upon arginine restoration.

Dispersed mammary gland epithelial cells. I. Isolation and separation procedures.

The mammary gland from midpregnant rabbits has been dissociated into individual cells by enzymatic digestion, divalent cation chelation, and gentle shearing. A heterogeneous cell population is obtained, comprising approximately 60% parenchymal cells, approximately 10% myoepithelial cells, and approximately 30% connective tissue cells, including fibroblasts, plasma cells, and microphages. The epithelial cells are characterized by the presence of fat droplets, which in 65% of the cells form large supranuclear vacuoles. Their buoyant density is less than 1.045, allowing their separation from myoepithelial cells and connective tissue cells by isopycnic centrifugation in a density gradient. The homogeneity of the epithelial cell fraction has been assessed by light and electron microscopy. The cells are viable and functionally active as indicated by their ability to exclude vital dyes, incorporate labeled precursors, consume oxygen, maintain intracellular Na+ and K+ concentrations, and retain their structural integrity. In addition, when cultured in Petri dishes, the cells grow as a monolayer, reestablish junctional complexes and retain cell polarity.