Integrative phylogenetic, phylogeographic and morphological characterisation of the Unio crassus species complex reveals cryptic diversity with important conservation implications.

The global decline of freshwater mussels and their crucial ecological services highlight the need to understand their phylogeny, phylogeography and patterns of genetic diversity to guide conservation efforts. Such knowledge is urgently needed for Unio crassus, a highly imperilled species originally widespread throughout Europe and southwest Asia. Recent studies have resurrected several species from synonymy based on mitochondrial data, revealing U. crassus to be a complex of cryptic species. To address long-standing taxonomic uncertainties hindering effective conservation, we integrate morphometric, phylogenetic, and phylogeographic analyses to examine species diversity within the U. crassus complex across its entire range. Phylogenetic analyses were performed using cytochrome c oxidase subunit I (815 specimens from 182 populations) and, for selected specimens, whole mitogenome sequences and Anchored Hybrid Enrichment (AHE) data on âˆ¼ 600 nuclear loci. Mito-nuclear discordance was detected, consistent with mitochondrial DNA gene flow between some species during the Pliocene and Pleistocene. Fossil-calibrated phylogenies based on AHE data support a Mediterranean origin for the U. crassus complex in the Early Miocene. The results of our integrative approach support 12 species in the group: the previously recognised Unio bruguierianus, Unio carneus, Unio crassus, Unio damascensis, Unio ionicus, Unio sesirmensis, and Unio tumidiformis, and the reinstatement of five nominal taxa: Unio desectusstat. rev., Unio gontieriistat. rev., Unio mardinensisstat. rev., Unio nanusstat. rev., and Unio vicariusstat. rev. Morphometric analyses of shell contours reveal important morphospace overlaps among these species, highlighting cryptic, but geographically structured, diversity. The distribution, taxonomy, phylogeography, and conservation of each species are succinctly described.

Effect of magnetic stimulation on the gene expression profile of in vitro cultured neural cells.

Transcranial magnetic stimulation is a non-invasive tool in clinical diagnostics and therapy for physiological and psychological diseases and has an increased application in experimental neurophysiology. Despite this, the mechanisms of magnetic stimulation of the central nervous system remain still unclear. We applied sinus-shaped high frequency magnetic fields in different stimulation patterns and repeated treatments to cell cultures derived from frontal cortex of murine embryos (BALB/cOlaHsd mice) to elucidate the effects of repetitive magnetic stimulation on the gene expression of in vitro cultured neural cells. Gene expression profiling was performed by using qRT-PCR array and single qRT-PCR analyses. Our methodological approach using microelectrode arrays data recording and analysis minimizes variations in transcriptome analysis arising from cell differentiation status and tissue complexity. With 10 significant changes in gene expression out of 171 genes using Alzheimer disease and neurodegeneration related qRT-PCR arrays we demonstrate significant impact of repetitive magnetic stimulation on the mRNA transcript of neural cell cultures. Sixteen candidate genes were analyzed using single qRT-PCR in a replicated statistical design, which provided more precise estimates of differences in expression profiles. We discussed the utility of the experimental methods used for cell culture selection and the changes in gene expression considering physiological aspects.

Impairments in pyridoxine-dependent sulphur amino acid metabolism are highly sensitive to the degree of vitamin B6 deficiency and repletion in the pig.

The objectives of the current study included the characterization of the temporal changes in indices of sulphur amino acid metabolism in piglets in response to vitamin B6 deficiency and repletion with graded levels of pyridoxine hydrochloride. In Experiment 1, 12 piglets (average initial weight = 5.3 kg; n = 6 per group) were fed a semi-purified diet containing either 0 (deficiency group) or 3 mg (control group) pyridoxine·HCl/kg diet, using a pair-feeding design, for 6 weeks. Piglets consuming vitamin B6-deficient diets exhibited decreased average daily gains on the 4th week and feed conversion efficiency from the 4th week until the end of the trial (P < 0.05). Plasma pyridoxal-5'-phosphate (PLP), in pigs consuming vitamin B6-deficient diets, was significantly lower than controls throughout the experiment (P < 0.01), reaching a nadir of 14% of the control animals' value by the end of the trial. Indices of sulphur amino acid metabolism, including activities of hepatic cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CGL) and serine hydroxymethyltransferase, as well as hepatic-free cysteine concentrations were markedly decreased after 6 weeks of B6 deficiency (P < 0.05). Total hepatic mRNA expressions for CBS and CGL were not affected. Concurrently, hepatic-free homocysteine concentrations increased by more than eight-fold (P < 0.01) at the end of the trial. An examination of plasma total homocysteine and cysteine concentrations revealed significant (P < 0.05) differences between treatments, with evidence of an abrupt shift in concentrations at 3 weeks post-initiation of dietary treatments (>25-fold increase in homocysteine; halving of cysteine values). At the end of Experiment 1, vitamin B6 deficiency significantly increased plasma methionine and serine levels, but decreased plasma glycine concentrations (P < 0.05). In Experiment 2, 20 pigs of 14 days old (initial BW = 5.0 kg) were subjected to a 4-week vitamin B6 depletion protocol, based on results obtained in Experiment 1. After the depletion period and assessment of baseline status (four pigs), remaining pigs were allocated to one of four dietary vitamin B6 repletion treatments: 0.75, 1.5, 2.25 and 3 mg/kg diet as pyridoxine·HCl (n = 4 per level) for 14 days. Significant dose-dependent increases in plasma PLP and cysteine, and decreases in homocysteine were observed, and these were sensitive to the duration of repletion. In conclusion, data from the current studies support the use of both plasma PLP and homocysteine as sensitive indices of vitamin B6 status in the pig. Additionally, the observed patterns of responses in vitamin B6-sensitive metabolites are supportive of an inclusion level of 2.25 mg/kg diet, as pyridoxine·HCl, in diets for young pigs.

The importance of genetic cluster recognition for the conservation of migratory fish species: the example of the endangered European huchen Hucho hucho (L.).

European huchen Hucho hucho (L.) is an endangered flagship species, which is endemic to the Danube drainage in central Europe. To date, no genetic information has been available as a basis for ongoing conservation and breeding programmes for the species. It is suspected that most populations in the wild share one common gene pool and that they exclusively depend on stocking with hatchery fish. In this study, highly variable microsatellite markers were established and the genetic diversity and differentiation from four important hatchery-reared stocks were compared with that of eight H. hucho populations sampled in the wild. Overall, eight genetic clusters with a moderate to very great degree of genetic differentiation and high assignment rates were identified. Each cluster contained individuals from two to 10 different populations and 9-100% of specimens from hatchery stocks. It is proposed that genetic cluster-based management in the conservation of European huchen is advantageous compared with the consideration of single local populations. A combined approach of maintaining the evolutionary potential of wild populations and genetically variable hatchery stocks can maximize the conservation of the species' evolutionary potential.

Mitochondrial and nuclear DNA phylogeography of European grayling (Thymallus thymallus): evidence for secondary contact zones in central Europe.

Mitochondrial and microsatellite DNA markers were applied to infer the phylogeography, intraspecific diversity and dynamics of the distributional history of European grayling (Thymallus thymallus) with focus on its central and northern European distribution range. Phylogenetic and nested clade analyses revealed at least four major mtDNA lineages, which evolved in geographical isolation during the Pleistocene. These lineages should be recognized as the basic evolutionary significant units (ESUs) for grayling in central and northern Europe. In addition, and in contrast to previous work on grayling, the results of Bayesian analysis of individual admixture coefficients, two-dimensional scaling analysis and spatial analysis of molecular variance provided evidence for a high level of admixture among major lineages in contact zones between drainages (e.g. the low mountain range of Germany), most likely resulting from glacial perturbations and ancient river connections between drainages during the Pleistocene glaciations. Even within river systems, a high level of differentiation among populations was revealed as indicated by the microsatellite data. Grayling sampled from 29 sites displayed high levels of differentiation (overall F(ST) = 0.367), a high number of private alleles and high bootstrap support for the genetic distance-based population clusters across 12 loci. We specifically discuss our results in context of phylogeograpic studies on other European freshwater fish species with habitat preferences similar to those of grayling. Our study shows that both large-scale phylogeographical and detailed genetic analyses on a fine scale are mandatory for developing appropriate conservation guidelines of endangered species.

Deletion of N-terminal rapsyn domains disrupts clustering and has dominant negative effects on clustering of full-length rapsyn.

The peripheral muscle membrane protein rapsyn is essential for the formation and maintenance of high density acetylcholine receptor aggregates at the neuromuscular synapse. Rapsyn is concentrated at synaptic sites and is colocalized with acetylcholine receptors from the earliest stages of synaptogenesis. Previous studies have shown that recombinant rapsyn expressed in heterologous cells forms clusters, and acetylcholine receptors coexpressed with rapsyn are colocalized with rapsyn clusters. However, the molecular interactions involved in clustering of rapsyn are not well defined. To analyze the process of cluster formation by rapsyn we examined the formation of rapsyn clusters and complexes using mutant constructs specifically deleted for individual domains of rapsyn in the presence and absence of tagged, full-length rapsyn. Specific deletions of the tetratricopeptide repeat (TPR) domains 1 and 3 of rapsyn abrogated not only clustering of mutant rapsyns, but also, in a dominant negative fashion, the clustering of tagged, full-length rapsyn. We also analyzed rapsyn protein complexes isolated from cells transfected with tagged and untagged rapsyn. Our results show that both tagged and untagged rapsyn are present in immunoprecipitates of rapsyn from cotransfected cells, demonstrating that rapsyn molecules interact directly or indirectly to form oligomers. Mutants that were dominant negatives were also present in complexes containing tagged, full-length rapsyn. Together these results indicate that rapsyn forms clusters at the synapse by oligomerization, and suggest models for the mechanistic bases of this oligomerization via interactions mediated by TPRs 1 and 3.

Genetic roots of the red deer (Cervus elaphus) population in Eastern Switzerland.

Overhunting of red deer (Cervus elaphus) in eastern Switzerland led to its extinction in the second half of the 17th century. Natural recolonization must have taken place later, because red deer were seen again in the canton of the Grisons (eastern Switzerland) in the 1870s. According to historical data, three different populations could have served as the source population. To determine the genetic origin of the eastern Swiss red deer population, we collected samples from five different subpopulations in the canton of the Grisons as well as from four adjacent populations in Germany, Liechtenstein, Austria, and Italy. We analyzed the samples by genotyping 18 microsatellite loci. F(ST) values, assignment tests, correspondence analysis, and fuzzy clustering clearly pointed to Liechtenstein as the most probable source population for the red deer in eastern Switzerland. In addition, our analyses revealed high gene diversity in all examined populations. Gene flow and the high genetic admixture are discussed.

Conditional deletion of brain-derived neurotrophic factor in the postnatal brain leads to obesity and hyperactivity.

Brain-derived neurotrophic factor has been associated previously with the regulation of food intake. To help elucidate the role of this neurotrophin in weight regulation, we have generated conditional mutants in which brain-derived neurotrophic factor has been eliminated from the brain after birth through the use of the cre-loxP recombination system. Brain-derived neurotrophic factor conditional mutants were hyperactive after exposure to stressors and had higher levels of anxiety when evaluated in the light/dark exploration test. They also had mature onset obesity characterized by a dramatic 80-150% increase in body weight, increased linear growth, and elevated serum levels of leptin, insulin, glucose, and cholesterol. In addition, the mutants had an abnormal starvation response and elevated basal levels of POMC, an anorexigenic factor and the precursor for alpha-MSH. Our results demonstrate that brain derived neurotrophic factor has an essential maintenance function in the regulation of anxiety-related behavior and in food intake through central mediators in both the basal and fasted state.

BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major components of plaque, beta-amyloid peptides (Abetas), are produced from amyloid precursor protein (APP) by the activity of beta- and gamma-secretases. beta-secretase activity cleaves APP to define the N-terminus of the Abeta1-x peptides and, therefore, has been a long- sought therapeutic target for treatment of AD. The gene encoding a beta-secretase for beta-site APP cleaving enzyme (BACE) was identified recently. However, it was not known whether BACE was the primary beta-secretase in mammalian brain nor whether inhibition of beta-secretase might have effects in mammals that would preclude its utility as a therapeutic target. In the work described herein, we generated two lines of BACE knockout mice and characterized them for pathology, beta-secretase activity and Abeta production. These mice appeared to develop normally and showed no consistent phenotypic differences from their wild-type littermates, including overall normal tissue morphology and brain histochemistry, normal blood and urine chemistries, normal blood-cell composition, and no overt behavioral and neuromuscular effects. Brain and primary cortical cultures from BACE knockout mice showed no detectable beta-secretase activity, and primary cortical cultures from BACE knockout mice produced much less Abeta from APP. The findings that BACE is the primary beta-secretase activity in brain and that loss of beta-secretase activity produces no profound phenotypic defects with a concomitant reduction in beta-amyloid peptide clearly indicate that BACE is an excellent therapeutic target for treatment of AD.

Angiogenesis, angiogenic growth factors, and cell adhesion molecules are upregulated in chronic pancreatic diseases: angiogenesis in chronic pancreatitis and in pancreatic cancer.

Recent epidemiologic evidence suggests that patients with chronic pancreatitis (CP) have an increased risk of developing pancreatic carcinoma (PCA). In spite of numerous similarities in both diseases, mechanisms for progression from CP to PCA are poorly understood. We hypothesized that enhanced angiogenesis might play a pivotal role in the etiology and histopathology of both CP and PCA, and thus form a possible link between precancer and carcinoma. In surgical specimens of 18 patients with CP, 10 with PCA, and 18 controls, absolute numbers of blood vessels and relative blood vessel density were assessed after immunostaining of endothelial cells for von Willebrand factor and PECAM-1 (platelet/ endothelial cell adhesion molecule-1). Furthermore, the expression of cell adhesion molecules ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) and of VEGF (vascular endothelial growth factor) was investigated in all specimens. Both CP and PCA exhibited areas of high vascular density ("hot spots"). The mean number of blood vessels in these areas in PCA was 132.2+/-16.8 per mm2, and in CP, 99.2+/-7.4 per mm2. The mean vessel count in controls was 25.1+/-5.1. Relative vessel density was increased in both PCA (41.3+/-3.5%) and CP (30.6+/-2.6%) versus controls (8.0+/-0.8%). Both absolute vessel count and relative vessel density were significantly higher (p<0.05) in PCA than in CP. Enhanced expression of ICAM-1 in CP and PCA was seen in ductal cells in CP and cancer cells. In controls, ICAM-1 and VCAM-1 were expressed only at low levels in endothelial cells. VCAM-1 was strongly expressed in acinar cells as well as in ductal cells. In CP and PCA, VEGF was strongly expressed in ductal cells in CP as well as in cancer cells. We show for the first time that angiogenic activity is increased in both CP and PCA. Based on this study, we suggest that antiangiogenesis might be a novel target for prevention or therapy in chronic pancreatic diseases.

Inhibition of bradykinin B2 receptor preserves microcirculation in experimental pancreatitis in rats.

The effect of B2 receptor bradykinin antagonist icatibant on postcapillary leukostasis, microcirculatory stasis, and tissue necrosis was studied in acute pancreatitis. In rats, pancreatitis was induced by intraductal injection of sodium taurocholate (ST), intravenous caerulein and intraductal infusion of glucodeoxycholic acid (GDOC), or intravenous caerulein infusion alone. Intravital pancreatic microcirculation was observed. Icatibant or vehicle was given 30 min before induction of pancreatitis. In ST pancreatitis, the number of perfused capillaries increased in icatibant-pretreated rats (77% vs. 0% for controls, P < 0.001). Capillary flow was preserved in icatibant-treated rats; total stasis was observed in controls. Mean venular leukocyte adherence decreased in icatibant-treated rats (26% vs. 74% for controls, P < 0.001), and median histopathologic score was reduced (icatibant vs. controls, 5.0 vs. 12 points, respectively; P < 0.01). Kinase II inhibitor captopril or exogenous bradykinin in addition to an otherwise effective dosage of icatibant resulted in microcirculatory stasis, extensive venular leukocyte adherence, and severe histological damage. With a 100 times greater icatibant dosage, this adverse effect was compensated. The beneficial effects of icatibant were also observed in intermediate pancreatitis (caerulein + GDOC). In ST and intermediate pancreatitis, icatibant preserved microcirculation, reduced venular leukocyte adherence, and prevented pancreatic tissue damage. B2 receptor bradykinin-mediated postcapillary leukostasis plays an important role in the pathogenesis of severe forms of acute pancreatitis.

Relationships among petroleum refining, water and sediment contamination, and fish health.

Water, sediment, and fish were sampled from three streams that were receiving or had received effluents from oil refineries. Water and sediment samples were analyzed by gas chromatography/mass spectrometry. Each stream contained aromatic carbons including substituted benzenes and naphthalenes, which are related to oil refinery operations. Fish were identified, counted, and examined for external lesions. Lengths and weights were recorded for older bullhead catfish, and their livers were examined histologically. Differences were seen in the diversity and abundance of fish among the upstream, impacted (effluent-receiving), and downstream stations. In one stream, differences in liver pathology were observed between reference bullhead, collected from an upstream station, and those collected at impacted stations with more than 50% of the bullheads taken from impacted stations having some sort of pathological change, including one with a liver clear-cell focus, which is considered a preneoplastic lesion in rodents. These data suggest a correlation between contamination of water and sediments with aromatic hydrocarbons, presumably from refinery effluents, and compromised fish health.

Pigmented subcutaneous spindle cell tumors in native gizzard shad (Dorosoma cepedianum).

An epizootic of pigmented subcutaneous spindle cell tumors affected nearly 25% of the adult gizzard shad (Dorosoma cepedianum) sampled from Lake of the Arbuckles in central Oklahoma over a 2 year period. Grossly, the tumors were primarily distributed over the head, trunk and fins as superficial raised masses that were almost always darkly pigmented. Histologically, they were located in the dermis, had a variable amount of connective tissue, and consisted of cells in a variety of forms and arrangements. Most tumors were composed of fusiform or spindle cells arranged in wavy bundles, whirling patterns or interwoven fascicles. Pigmentation was attributed to large dense deposits of melanin or to scattered individual melanin-containing cells. Immunohistochemical detection of proliferating cell nuclear antigen revealed a high proliferative activity in the spindle cells. Electron microscopy showed that the tumors were composed of several cell types, including host reactive cells, melanocytes in stages of maturity, and fibroblast-like cells. Tumor cells had neither cell-to-cell junctions nor an external lamina. Although the cell of origin of the tumors was not identified, evidence points toward melanocytes or, possibly, nerve sheath cells. However, an origin from fibroblasts or some other poorly differentiated cell cannot be ruled out. The etiology of the tumors was not determined. Fractionation of lake water and sediment samples followed by GC-MS analysis revealed no carcinogenic compounds. A retroviral etiology is unlikely because assays for reverse transcriptase in tumor homogenates were negative, and no evidence of viral particles was found in specimens examined by electron microscopy.

Differences in the frequency of cholesterol screening in patients with Medicaid compared with private insurance.

OBJECTIVE: To assess compliance with preventive screening for Medicaid recipients compared with individuals with equal access to preventive services. SETTING: A community-based family practice residency program. METHODS: Survey of a consecutive sample of English-reading individuals, aged 18 to 50 years, with Medicaid (n = 98) or private insurance (n = 75), who had scheduled appointments in the clinic. MAIN RESULTS: Patients with Medicaid were as likely as those with private insurance to be screened for hypertension and cervical cancer in the last 5 years but were less likely to have received cholesterol screening (39% vs 65%, P < .001). Even after adjusting for differences in gender composition, age, race, marital status, and education level attained, patients with Medicaid were still less likely to have received cholesterol screening (odds ratio, 0.43; 95% confidence interval, 0.21 to 0.57). Although patients with Medicaid were no more likely to identify a barrier and, when identifying barriers, did not identify significantly more barriers than patients with private insurance, Medicaid recipients were less likely to state that cholesterol testing had been recommended by their physician (30% vs 44%, P = .05). CONCLUSIONS: Because access to screening tests by patients with Medicaid is equal to or better than that of those with insurance, differences in the frequency of cholesterol screening should not reflect financial barriers. Differences in the attention paid to screening by physicians and differences in cultural beliefs about the importance of screening may play a role in the underuse of screening services by individuals in lower socioeconomic groups.

The effect of albumin or crystalloid resuscitation on bacterial translocation and endotoxin absorption following experimental burn injury.

Burn injury induces immune suppression and increases susceptibility to infection. Hypoalbuminemia is an early and consistent finding following thermal injury and is independently associated with gastrointestinal dysfunction and increased rates of infectious morbidity. This study assessed the effects of albumin resuscitation on burn-induced immunosuppression, bacterial translocation, and absorption of gut endotoxin. Male Sprague-Dawley rats, presensitized to keyhole limpet hemocyanin (KLH), underwent a 20% dorsal scald burn injury, followed by laparotomy and IVC catheterization for fluid resuscitation. Animals were randomized to one of three resuscitative regimens: Ringer's lactate 3 ml/kg/% burn, Ringer's lactate 9 ml/kg/% burn, or 5% human albumin 3 ml/kg/% burn. Delayed hypersensitivity (DTH) responses to KLH were depressed 24 hr following injury (preburn 8.9 +/- 0.2 mm, post-burn 3.1 +/- 0.3 mm, P less than 0.001) and were significantly lower in animals in whom gram-negative bacterial translocation had occurred (2.3 +/- 0.4 vs 3.6 +/- 0.2 mm, P less than 0.005). Serum albumin levels were lower and rates of gram-negative bacterial translocation higher for those animals receiving low volume crystalloid resuscitation; animals resuscitated with albumin or high volume crystalloid experienced similar degrees of postinjury hypoalbuminemia and bacterial translocation. Uptake of radiolabeled endotoxin was maximal in animals resuscitated with albumin. Bacterial translocation is believed to be responsible for a significant number of late nosocomial infections following trauma. These data suggest that the adequacy of early resuscitation rather than the type of resuscitative solution is the more important factor in minimizing translocation.

Comparison of the distributions of D1 and D2 dopamine receptor mRNAs in rat brain.

The distributions of messenger RNAs encoding both the D1 and D2 dopamine receptors have been determined in the rat brain by in situ hybridization. High levels of both mRNAs were found in the traditional dopaminoceptive regions of brain, including the caudate-putamen, nucleus accumbens, and olfactory tubercle; lower levels of both were found in a number of other neural structures, such as the lateral septum, olfactory bulb, hypothalamus, and cortex. High levels of D2 but not D1 receptor mRNA were identified in the midbrain dopamine cell groups, suggesting that the autoreceptors found in the substantia nigra and ventral tegmental area are exclusively D2. Other areas demonstrating differential distribution of these two mRNAs included the pituitary, amygdala, and hippocampus. Quantitative densitometric analysis revealed that in most of the brain regions studied in which both messages exist, the amounts of D1 and D2 receptor mRNAs were approximately equal. Finally, using thin (2.5-micron) sections through the caudate-putamen, about half of all cells were found to be positive for D1 receptor mRNA, and approximately 75% of cells contained D2 receptor mRNA. Subsequent analysis in sequential sections revealed that co-localization of D1 and D2 receptor mRNA occurred in 33% +/- 7% of all caudate-putamen cells: about half of all cells containing D1 receptor mRNA also contained D2 receptor mRNA, and approximately half of all D2 receptor mRNA-positive cells also contained D1 receptor mRNA. These results indicate that there is considerable overlap between D1 and D2 dopaminoceptive cells, and provide a basis for future regulatory studies of dopamine systems in brain within a defined anatomic context.

Methods for determination of growth-rate-dependent changes in hybridoma volume, shape and surface structure during continuous recycle.

Hybridoma volume and surface membrane structure were found to vary as a function of specific growth rate using a method of cell recycle with continuous medium perfusion to vary growth rate. Mean hybridoma volume determined at constant osmolality by both electronic particle counting and scanning electron microscopic (SEM) methods indicated that rapidly growing cells are significantly larger than very slowly growing cells. We have previously determined that during both rapid and slow growth over a range of L-glutamine provision rates (Gln PR) that specific monoclonal antibody (MoAb) secretion rate was not changed. In this study a constant MoAb secretion rate per unit of membrane area was found which may indicate that changing membrane area is not a rate-determining factor in MoAb secretion. SEM methods were of limited use for accurate determination of cell volume due to cell shrinkage and large coefficients of variations. In spite of this limitation, SEM stereology methods were useful in confirming that cells remained spherical over a wide range of specific growth rates and that hybridoma cells were not circular. Sequential SEM observations also revealed that surface membrane structure of the 9.2.27 murine hybridoma investigated was correlated with growth rate. Under conditions of very slow growth, hybridoma surface microvilli density appeared to be significantly reduced.

Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning electron microscopy.

The higher-order assembly of the approximately 30 nm chromatin fibers into the characteristic morphology of HeLa mitotic chromosomes was investigated by electron microscopy. Transmission electron microscopy (TEM) of serial sections was applied to view the distribution of the DNA-histone-nonhistone fibers through the chromatid arms. Scanning electron microscopy (SEM) provided a complementary technique allowing the surface arrangement of the fibers to be observed. The approach with both procedures was to swell the chromosomes slightly, without extracting proteins, so that the densely-packed chromatin fibers were separated. The degree of expansion of the chromosomes was controlled by adjusting the concentration of divalent cations (Mg2+). With TEM, individual fibers could be resolved by decreasing the Mg2+ concentration to 1.0-1.5 mM. The predominant mode of fiber organization was seen to be radial for both longitudinal and transverse sections. Using SEM, surface protuberances with an average diameter of 69 nm became visible after the Mg2+ concentration was reduced to 1.5 mM. The knobby surface appearance was a variable feature, because the average diameter decreased when the divalent cation concentration was further reduced. The surface projections appear to represent the peripheral tips of radial chromatin loops. These TEM and SEM observations support a "radial loop" model for the organization of the chromatin fibers in metaphase chromosomes.