The membrane-associated enzyme phosphatidylserine synthase is regulated at the level of mRNA abundance.

To precisely define the functional sequence of the CHO1 gene from Saccharomyces cerevisiae, encoding the regulated membrane-associated enzyme phosphatidylserine synthase (PSS), we subcloned the original 4.5-kilobase (kb) CHO1 clone. In this report a 2.8-kb subclone was shown to complement the ethanolamine-choline auxotrophy and to repair the defect in the synthesis of phosphatidylserine, both of which are characteristic of cho1 mutants. When this subclone was used as a hybridization probe of Northern and slot blots of RNA from wild-type cells, the abundance of a 1.2-kb RNA changed in response to alterations in the levels of the soluble phospholipid precursors inositol and choline. The addition of inositol led to a 40% repression of the 1.2-kb RNA level, while the addition of choline and inositol led to an 85% repression. Choline alone had little repressive effect. The level of 1.2-kb RNA closely paralleled the level of PSS activity found in the same cells as determined by enzyme assays. Disruption of the CHO1 gene resulted in the simultaneous disappearance of 1.2-kb RNA and PSS activity. Cells bearing the ino2 or ino4 regulatory mutations, which exhibit constitutively repressed levels of a number of phospholipid biosynthetic enzymes, had constitutively repressed levels of 1.2-kb RNA and PSS activity. Another regulatory mutation, opi1, which causes the constitutive derepression of PSS and other phospholipid biosynthetic enzymes, caused the constitutive derepression of the 1.2-kb RNA. When cho1 mutant cells were transformed with the 2.8-kb subclone on a single-copy plasmid, the 1.2-kb RNA and PSS activity levels were regulated in a wild-type fashion. The presence of the 2.8-kb subclone on a multicopy plasmid, however, led to the constitutive overproduction of 1.2-kb RNA and PSS in cho1 cells.     

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.