Isolation and culture of cells derived from human cerebral microvessels

Summary Microvessels were isolated from non-neoplastic human cerebral cortical fragments resected for treatment of intractable seizure disorder. The microvessels were incubated in modified Lewis medium with 20 or 30% fetal bovine serum. Within 1–2 weeks, two cell populations emerged from the isolates. One type of cells had polygonal morphology, showed density-dependent contact inhibition at confluence in vitro, showed lectin-binding characteristics of endothelium (but only moderate positivity for factor VIII antigen), demonstrated induction of -glutamyl trans-peptidase when exposed to astrocyte-conditioned media, and responded to insulin by a pronounced increase in DNA synthesis. The other variety of cells grew in vitro more slowly in irregular strands separated by clear zones, showed ultrastructural features of smooth muscle, and isoelectric focusing of cell proteins revealed the presence of smooth-musclespecific -isoactin. Both types of cells could be serially subcultured. The ability to isolate and grow the two cell types, tentatively identified as human cerebral microvascular endothelium and smooth muscle, may facilitate studies of human blood-brain barrier function as well as the pathogenesis of cerebral microangiopathies unique to the human brain.Funded by Canadian Heart Foundation, Heart and Stroke Foundation of Ontario and UCLA Biomedical Research Support Grant     

The human kappa deleting element and the mouse recombining segment share DNA sequence homology.

We have used cloned mouse and human DNA probes to identify regions of conserved homology between the human and murine DNA segments, (termed kappa deleting element (kde) and recombining segment (RS) respectively) which are frequently recombined in lambda-producing B cells. Heteroduplex analysis indicated extensive homology in the region immediately downstream of the recombination site of both segments. This was confirmed by Southern and direct nucleotide sequence analyses. Fifty percent homology was detected within the 500 nucleotides that neighbour the recombination points in the kde and RS segments. These results indicate that the kde and RS sequences are evolutionarily conserved and may be functionally relevant to normal B cell development.     

Neural control of a cyclic postural behavior in the crayfish,Procambarus clarkii: the pattern-initiating interneurons

As part of its repertoire of defensive behaviors, the crayfish, Procambarus clarkii, may respond to mildly threatening tactile or visual stimuli from the front of its body by walking backwards. During this behavior, the abdomen undergoes complex cyclical movements involving flexion and extension of the postural musculature which cause the tail to alternately contact and withdraw from the substrate. Intracellular neuropil recordings and dye injections were used to search for the interneurons responsible for initiating this postural motor pattern in the crayfish abdomen. Several diverse morphological types of interganglionic pattern-initiating (PI) interneurons were found. Each interneuron, when driven intracellularly, was capable of eliciting the same motor program, in its entirety, throughout the abdominal nerve cord. During pattern generation, PI interneurons exhibited a burst of spikes preceding the motor output. Silencing single PI interneurons with hyperpolarizing current during pattern generation failed to affect the motor program, indicating a redundancy of pattern-initiating function. The observations of extensive dye-coupling with other parallel axons, consistent dye-coupling with other identified cells in the pattern-initiating system, and the presence of multiple spike amplitudes in the bursts suggested electrotonic coupling among the PI interneurons. An additional group of interganglionic interneurons, the partial pattern-initiating (PPI) interneurons, were found to comprise a significant subset of the pattern-initiating system. As with the PI cells, the PPI interneurons exhibited a complex burst of spikes just preceding the patterned motor program. However, the PPI interneurons were only capable of eliciting an incomplete, though recognizable, postural motor pattern. Silencing any PPI interneuron during pattern generation caused a deficit in the motor pattern, indicating either an absence or lesser degree of functional redundancy within the PPI interneuron population compared to that occurring within the PI interneuron group. We conclude that a large number of PI interneurons are presynaptic to a relatively small group of PPI interneurons which, in turn, conduct pattern-initiating signals to the ganglionic oscillators. Our results indicate that pattern-initiation is accomplished through a command system involving multiple command elements organized in a coordinated interganglionic network.     

Role of nonohmicity in the regulation of electron transport in plant mitochondria

The relationship between the respiratory rate and the membrane ionic current on the protonmotive force has been investigated in percoll purified potato mitochondria. The dependence of the membrane ionic current on the membrane potential was monitored using a methyltriphenylphosphonium-sensitive electrode and determining the maximal net rate of depolarization following the addition of a respiratory inhibitor. We have confirmed that a nonohmic relationship exists between the ionic conductance and membrane potential. Addition of ATPase inhibitors markedly increased the initial rate of dissipation suggesting that in their absence the dissipation rate induced by respiratory inhibitors is partially offset by H⁺-efflux due to the hydrolysis of endogenous ATP. This was corroborated by direct measurement of endogenous ATP levels which decreased significantly following dissipation of the membrane potential. Results are discussed in terms of the regulation of electron transport in plant mitochondria in vivo.     

The response of murine intestinal crypts to short-range promethium-147 beta irradiation: deductions concerning clonogenic cell numbers and positions

An exteriorized loop of mouse intestine was exposed to 147Pm low-energy electrons, where the dose rate decreased by a factor of 5 from the base of the crypt to the top of the proliferative zone. A crypt survival curve was obtained, expressed in terms of exposure time. The shape of the curve was interpreted in terms of survival parameters for colony-forming cells (clonogens) derived using 137Cs gamma rays and the depth-dose curve measured for 147Pm electrons. It is concluded that the shape of the crypt survival curve using 147Pm electrons is inconsistent with the notion of either the presence of a large number of clonogens or a small number near the top of the proliferative zone. A computer fitting procedure showed that the best agreement between predicted and observed curves was achieved with 2.7 +/- 0.5 clonogens at cell position 5.6 +/- 0.6, in the putative stem-cell zone.     

Mucosal epithelial cells and Langerhans cells are targets for infection by the immunosuppressive type D retrovirus simian AIDS retrovirus serotype 1

Simian acquired immune deficiency syndrome (SAIDS) caused by the type D retrovirus SRV-1 results in opportunistic infections and a spectrum of oral lesions similar to those seen in humans with AIDS. To better understand the pathogenesis of these oral lesions we have retrospectively examined the oral mucosa from ten rhesus monkeys that died with SAIDS and prospectively examined the oral mucosa of ten additional animals inoculated with SRV-1 to determine at what time, and in what cells SRV-1 infection of the oral mucosa occurs. Using single and double label immunohistologic techniques, and electron microscopy we detected SRV-1 in clusters of oral epithelial cells and rare Langerhans cells as early as 1 month postinoculation.     

Herpes simplex virus-1-specific proteins are involved in alteration of polyphosphoinositide metabolism in baby-hamster kidney cells.

Herpes simplex virus type 1 (HSV-1) induces altered phosphoinositide metabolism in baby hamster kidney (BHK) cells, measured as incorporation of [3H]inositol or [32P]Pi [Langeland, Haarr & Holmsen (1986) Biochem. J. 237, 707-712]. We now report that this response in the inositol phospholipids is dependent on virus-specific proteins synthesized in the beta (early) stage of virus protein synthesis. This was demonstrated both by resistance to the inhibitory effect of cycloheximide after this stage of infection, and by the use of temperature-sensitive (ts) mutants of HSV-1; ts mutants in which protein synthesis was blocked so that only the alpha proteins were expressed showed a PIP2/PIP (phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 4-monophosphate) ratio similar to uninfected cells, while ts mutants which were defective in protein synthesis at a late beta stage or later showed increased PIP2/PIP ratios similar to cells infected by wild type HSV-1.     

SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA.

We have reconstituted protein translocation across plasma membrane vesicles of Escherichia coli using purified proOmpA and trigger factor, a 63 kd soluble protein. Treatment of membrane vesicles with urea inactivates them for translocation unless a factor present in cytoplasmic extracts is added during the translocation reaction. Sedimentation analysis showed that the stimulatory activity is of distinctly higher mol. wt than trigger factor. Cytoplasmic extracts from a strain that greatly overproduces the SecA protein are highly enriched in the stimulatory activity for untreated membranes and restore translocation to urea-treated membranes, suggesting that this protein is the stimulatory factor. This assay was used to monitor the isolation of SecA protein from the overproducing strain. The purified protein is soluble, yet binds peripherally to membranes with high affinity and supports translocation. Using pure proOmpA, SecA protein, trigger factor and urea-treated membranes, the protein export process was resolved into binding and translocation steps. We find that proOmpA binds to membrane vesicles with or without SecA protein, but that translocation only occurs when SecA was bound prior to proOmpA.     

Linkage analysis of chromosome 17 markers in British and South African families with neurofibromatosis type 1

Nine markers from the pericentromeric region of chromosome 17 were typed in 16 British and five South African families with neurofibromatosis type 1 (NF1). The markers--p17H8, pHHH202, and EW204--were linked to NF1 at recombination fractions less than 1%. No evidence of locus heterogeneity was detected. Inspection of recombinant events in families informative for several markers suggests that the NF1 gene is located between the markers EW301 (cen-p11.2) and EW206 (cen-q12) and possibly distal to pHHH202 (q11.2-q12).