Parametric investigation of static and dynamic cell culture conditions and their impact on hCMEC/D3 barrier properties.

In brain research, the hCMEC/D3 cell line is widely used for the establishment of a human in vitro blood-brain barrier (BBB) model. However, its barrier integrity seems to be insufficient for drug permeability studies, represented by rather low transendothelial electrical resistance (TEER) and high permeability of small molecules. Therefore, this study covers a parametric investigation of static and dynamic cell culture conditions to improve barrier functionality of hCMEC/D3. The effect of basal media was investigated by analyzing changes in proliferation rate, barrier integrity and gene expression of cellular junction proteins. The cells were able to grow in different cell culture media, including serum-free media. However, none of these media enhanced strongly the growth rate or barrier integrity compared to the microvascular endothelial cell growth medium-2 (EGM™-2 MV). Furthermore, hCMEC/D3 cells did not respond positively regarding TEER to any tested parameter neither supplements, coating materials nor co-cultures with the human immortalized astrocyte cell line SVGmm. Furthermore, the impact of dynamic conditions was examined by using the Dynamic Micro Tissue Engineering System (DynaMiTES). Cultivation conditions were successfully adapted to the DynaMiTES design and no negative effect was detected by analyzing cell viability and cell count, albeit TEER remained also unchanged. Consequently, the hCMEC/D3 model has considerable limitations and further improvements or alternative cell lines are required.

Dicondylar humeral fracture stabilisation in a dog using a transilial rod and external fixation.

Repair of a Salter-Harris type IV dicondylar humeral fracture was performed on a 15-week-old pitbull terrier. Interfragmentary compression of the intracondylar component of the fracture was achieved with a transilial rod and locking nuts (Trans-ilial Rod; IMEX Veterinary, Inc.). The transilial rod was articulated with a modified type I external fixator which functioned as adjunctive stabilisation for the supracondylar component of the fracture. Fracture healing was confirmed radiographically five weeks following surgery. The dog had no appreciable lameness when examined 12 months after fracture repair.

Anesthetic and cardiorespiratory effects of a 1:1 mixture of propofol and thiopental sodium in dogs.

OBJECTIVE: To compare anesthetic and cardiorespiratory effects of a 1:1 (vol:vol) mixture of propofol and thiopental sodium with either drug used alone in dogs. DESIGN: Randomized crossover study. ANIMALS: 10 healthy Walker Hounds. PROCEDURE: Dogs received propofol (6 mg/kg [2.7 mg/lb] of body weight), thiopental (15 mg/kg [6.8 mg/lb]), or a mixture of propofol (6 mg/kg) and thiopental (15 mg/kg) at 1-week intervals. Drugs were slowly administered i.v. over 90 seconds or until dogs lost consciousness. Increments of 10% of the initial dose were administered until intubation was possible. Amount of drug required for intubation, quality of induction and recovery, times from induction to intubation and to walking with minimal ataxia, and duration of intubation and lateral recumbency were recorded. Heart and respiratory rates, mean, systolic, and diastolic blood pressure, hemoglobin saturation of oxygen (SpO2), and end-tidal CO2 concentration (ETCO2) were determined before and after intubation. RESULTS: Amounts of propofol and thiopental required to permit intubation were less, but not significantly so, when administered in combination than when administered alone. Duration of lateral recumbency and time from induction to walking were greater and recovery quality was worse in the thiopental group, compared with the other groups. Dogs in all groups remained normotensive. Respiratory rate, heart rate, ETCO2, and SpO2 did not differ among groups. CONCLUSIONS AND CLINICAL RELEVANCE: A 1:1 mixture of propofol and thiopental induced anesthesia of similar quality to propofol or thiopental alone. Recovery quality and recovery times were similar to those of propofol and superior to those of thiopental.

Spatial organization of four hnRNP proteins in relation to sites of transcription, to nuclear speckles, and to each other in interphase nuclei and nuclear matrices of HeLa cells.

RNA polymerase II transcripts are complexed with heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. These proteins are involved in several aspects of the maturation and transport of hnRNA. We performed a detailed study of the spatial distribution of four hnRNP proteins (hnRNP C, I, L, and U) in HeLa nuclei, using immunofluorescent labeling and confocal microscopy. Despite the fact that hnRNP proteins have been shown to coimmunoprecipitate, a hallmark of hnRNP proteins, we find that hnRNP C, I, and L have a spatial nuclear distribution that is not related to that of hnRNP U. We also examined the distribution of hnRNP proteins in relation to that of nascent transcripts. The four hnRNP proteins that we examined are not enriched at sites of RNA synthesis. Using antibodies against the nuclear poly(A)-binding protein (PAB II) we investigated the relationship between the distribution of hnRNP proteins and that of nuclear domains (nuclear speckles) that are enriched in splicing factors, poly(A)+RNA, and PAB II. We found that the four hnRNP proteins are not enriched in these domains. This indicates that the poly(A)+RNA, present in high concentration in speckles, is not complexed with these hnRNP proteins. This is in agreement with the notion that poly(A)+RNA in speckles is different from ordinary hnRNA. Previously, we have shown that hnRNP proteins are the major protein components of the fibrogranular internal nuclear matrix (K. A. Mattern et al. (1996) J. Cell. Biochem. 62, 275-289; K. A. Mattern et al. (1997) J. Cell. Biochem. 65, 42-52). We observed that in nuclear matrices the spatial distributions of the four hnRNP proteins, like that of nascent RNA and PAB II, are essentially the same as observed in intact nuclei. Moreover, also in nuclear matrix preparations, like in intact nuclei, nascent RNA and PAB II have spatial distributions that differ from those of hnRNP proteins. Our results are compatible with the notion that hnRNP proteins are able to form complexes of many different, probably overlapping, compositions.

Major internal nuclear matrix proteins are common to different human cell types.

The nuclear matrix may be involved in the structural and functional organization of the cell nucleus. However, we still do not understand the molecular basis of the intranuclear fibrogranular network that is part of the nuclear matrix. We recently described a method to identify internal nuclear matrix proteins [Mattern et al. (1996): J Cell Biochem 62:275-289], which was done by comparing two nuclear matrix preparations: one with and one without the internal structure by using quantitative two-dimensional gel electrophoresis. In the present study, we use the same approach to compare the nuclear matrix proteins of four different human cell types to investigate whether they have a similar internal nuclear matrix protein composition. Major nuclear matrix proteins present in all these cell types likely represent the base of the internal nuclear matrix. We demonstrate that the 25 most abundant internal nuclear matrix proteins are common to all four cell types. Together, these common proteins represent more than 75% of the total internal nuclear matrix protein mass in each cell type. This set of proteins includes B23 and most hnRNP proteins. The quantity of most of these proteins is very similar in the four cell types. The fact that the internal nuclear matrix consists mainly of hnRNP proteins, which may be involved in transcription, transport, and processing of hnRNA, supports the idea that the internal nuclear matrix is the result of these processes.

hnRNP proteins and B23 are the major proteins of the internal nuclear matrix of HeLa S3 cells.

The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina-pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa 53 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high-resolution two-dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA.

Nuclear domains involved in RNA synthesis, RNA processing, and replication.

Two main principles of nuclear organization have been outlined on the basis of contributions by many research groups in recent years. The first principle is that interphase chromosomes occupy discrete territories in the nucleus, with no intermingling of the DNA from different chromosomes. Within a chromosome territory the DNA is organized in chromatin fibers at several levels of folding, that meander through the territory. Transcription and replication take place at the surface of these higher order chromatin fibers, probably on locally unfolded DNA templates. The second principle is that different types of nuclear domains are associated with several specific gene loci. This holds for clusters of interchromatin granules, coiled bodies, RNA 3'-cleavage factor-containing nuclear bodies (cleavage bodies) and probably PML-containing nuclear bodies. These domains may play an important role in the spatial arrangement of genes in the interphase nucleus. Despite these new insights, our knowledge of the function of many nuclear compartments and the molecular interactions responsible for the dynamic organization of a compartmentalized nucleus is still in its infancy.

The aerobic growth of Propionibacterium acnes in primary cultures from skin.

Although Propionibacterium acnes ordinarily grows as an anaerobe, this organism was commonly seen on aerobic primary cultures (directly from skin) of the forehead, axilla, deltoid area, and antecubital fossa. On subculture, strains from aerobic primary cultures showed their normal anaerobic state. On repeated tests of the foreheads of 7 subjects over periods of 1 to 4 yr, 24% of all cultures were positive for propionibacteria (P. acnes) on aerobic culture. This phenomenon was equally common in cultures from those with relatively sparse or dense populations of P. acnes on the skin surface. Several different patterns of aerobic growth were observed: satellitism, profuse growth at one dilution and none on the next higher dilution, and proportionate growth in successive dilutions. These different growth patterns indicate that more than one mechanism is operative in stimulating the aerobic growth of propionibacteria. Whether one or more of these mechanisms is operative in vivo, we do not know, but it appears that they do not account for the great individual differences in population density of P. acnes on such sites as the forehead.

Selective antibacterial action of 2-mercaptoethanol on propionibacteria in skin cultures.

2-Mercaptoethanol applied to the surface of agar medium had a selective antibacterial effect on Propionibacterium acnes and Propionibacterium granulosum without interfering with the growth of Peptococcus saccharolyticus or staphylococci in anaerobic cultures of skin or in pure cultures. In aerobic broth culture, 2-mercaptoethanol inhibited aerobes and stimulated anaerobes, consistent with its action as a reducing agent.

Individual differences in the bacterial flora of the skin of the forehead: Peptococcus saccharolyticus.

Peptococcus saccharolyticus was a numerically important constituent of the bacterial flora of the forehead of 20% of 40 subjects. 16 of these subjects were studied over periods of 12 to 54 mo. It was consistently absent from 12 subjects, was present on every test of 3 subjects, and constituted 96 to 100% of the total flora on 1 subject tested 8 times in a 16-month period. On the forehead of one subject, Peptococcus saccharolyticus was recovered in only 1 of 5 tests.

Isolation and identification of Peptococcus saccharolyticus from human skin.

Peptococcus saccharolyticus was a numerically important organism on the forehead and the antecubital fossa of the arm of ca. 20% of the subjects studied. It was best detected by prolonged (4- to 7-day) anaerobic incubation on a suitably enriched medium but also appeared in smaller numbers in aerobic primary cultures after 4 to 7 days. Initial screening was based on growth patterns in shake cultures and on the relative amounts of growth on aerobic and anaerobic streak plates. The organism was nonhemolytic and usually catalase positive. It produced acid anaerobically from glucose, fructose, and glycerol, but not from maltose, and did not produce lactic acid from glucose.