A New Pressure Probe Method to Determine the Average Volumetric Elastic Modulus of Cells in Plant Tissue

A new in vivo method was used to determine an average volumetric elastic modulus ([epsilon]ave) for nongrowing cells in plant tissue. This method requires that both the relative transpiration rate, T, of the tissue and the average turgor pressure decay rate, (dP/dt)ave, of the cells are measured after the water source is removed from the plant tissue. Then [epsilon]ave is calculated from the equation [epsilon]ave = (-dP/dt)ave/T. This method was used to determine [epsilon]ave for cortical cells in stems of pea seedlings (Pisum sativum L.). The results demonstrate that [epsilon]ave increases from virtually zero at low P (approximately 0.01MPa) to approximately 10 MPa at high P (approximately 0.5 MPa). Analyses of the results indicate that the relationship between [epsilon]ave and P can be approximated by a linear function and more accurately approximated by a saturating exponential function: [epsilon]ave = [epsilon][infinity symbol][1 - exp {-k(P - Po)}], where Po is a plateau pressure (approximately 0.01 MPa), k is a rate constant (approximately 7 per MPa), and [epsilon][infinity symbol] (approximately 10 MPa) is the hypothetical maximum value of [epsilon]ave as P -> [infinity symbol]. Solutions for the turgor pressure decay (due to transpiration) as functions of time and symplasmic water mass (after the water source is removed) are derived.     

Identification of Trans-Acting Genes Necessary for Centromere Function in Drosophila Melanogaster Using Centromere-Defective Minichromosomes

Deletions in the Drosophila minichromosome Dp1187 were used to investigate the genetic interactions of trans-acting genes with the centromere. Mutations in several genes known to have a role in chromosome inheritance were shown to have dominant effects on the stability of minichromosomes with partially defective centromeres. Heterozygous mutations in the ncd and klp3A kinesin-like protein genes strongly reduced the transmission of minichromosomes missing portions of the genetically defined centromere, but had little effect on the transmission of minichromosomes with intact centromeres. Using this approach, ncd and klp3A were shown to require only the centromeric region of the chromosome for their roles in chromosome segregation. Increased gene dosage also affected minichromosome transmission and was used to demonstrate that the nod kinesin-like protein gene interacts genetically with the centromere, in addition to interacting with extracentromeric regions as demonstrated previously. The results presented in this study strongly suggest that dominant genetic interactions between mutations and centromere-defective minichromosomes could be used effectively to identify novel genes necessary for centromere function.     

Identification of a Novel Stage of Ribosome/Nascent Chain Association with the Endoplasmic Reticulum Membrane

Protein translocation in the mammalian endoplasmic reticulum (ER) occurs cotranslationally and requires the binding of translationally active ribosomes to components of the ER membrane. Three candidate ribosome receptors, p180, p34, and Sec61p, have been identified in binding studies with inactive ribosomes, suggesting that ribosome binding is mediated through a receptor-ligand interaction. To determine if the binding of nascent chain-bearing ribosomes is regulated in a manner similar to inactive ribosomes, we have investigated the ribosome/nascent chain binding event that accompanies targeting. In agreement with previous reports, indicating that Sec61p displays the majority of the ER ribosome binding activity, we observed that Sec61p is shielded from proteolytic digestion by native, bound ribosomes. The binding of active, nascent chain bearing ribosomes to the ER membrane is, however, insensitive to the ribosome occupancy state of Sec61p. To determine if additional, Sec61p independent, stages of the ribosome binding reaction could be identified, ribosome/nascent chain binding was assayed as a function of RM concentration. At limiting RM concentrations, a protease resistant ribosome-membrane junction was formed, yet the nascent chain was salt extractable and cross-linked to Sec61p with low efficiency. At nonlimiting RM concentrations, bound nascent chains were protease and salt resistant and cross-linked to Sec61p with higher efficiency. On the basis of these and other data, we propose that ribosome binding to the ER membrane is a multi-stage process comprised of an initial, Sec61p independent binding event, which precedes association of the ribosome/nascent chain complex with Sec61p.     

The temperature-sensitive (ts) phenotype of a cold-passaged (cp) live attenuated respiratory syncytial virus vaccine candidate, designated cpts530, results from a single amino acid substitution in the L protein.

cpts530, a candidate live-virus vaccine, is an attenuated strain of human respiratory syncytial virus (RSV). It was derived by subjecting a cold-passaged (cp) strain of RSV to a single round of chemical mutagenesis. cpts530 is a temperature-sensitive (ts) mutant that is attenuated in mice and chimpanzees, and its ts phenotype exhibits a high level of stability during replication in both species. In the present study, the complete nucleotide sequence of cpts530 RSV was determined. The five mutations known to be present in the parent cpRSV were retained in its cpts530 derivative, and one additional nucleotide change was identified at nucleotide (nt) 10060, which resulted in a phenylalanine-to-leucine change at amino acid 521 in the large polymerase (L) protein. To determine if this single amino acid substitution was indeed responsible for the ts phenotype of cpts530, it was introduced alone or in combination with the cp mutations into the full-length cDNA clone of the wild-type A2 RSV. Analysis of infectious viruses recovered from mutant cDNAs indicated that this single mutation specified complete restriction of plaque formation of recombinant cp530 in HEp-2 cell monolayer cultures at 40 degrees C, and the level of temperature sensitivity was not influenced by the presence of the five cpRSV mutations. These findings identify the phenylalanine-to-leucine change at amino acid 521 in the L protein as the mutation that specifies the ts phenotype of cpts530. Furthermore, these findings illustrate the feasibility of using the cDNA-based recovery system to analyze and construct defined attenuated vaccine viruses.     

Crystallization and X-ray analysis of a single fab binding domain from protein L of Peptostreptococcus magnus

Protein L is a multi domain cell wall constituent of certain strains of Peptostreptococcus magnus which binds to the variable domain of immunoglobulin κ-light chains. A single immunoglobulin-binding domain of M_r = 9000 from this protein has been isolated and crystallized. The crystals are of space group P4₂2₁2, with cell dimensions a = b = 66.9 Å, c = 68.3 Å, and diffract to at least 2.2 Å resolution. The asymmetric unit of the crystal contains two molecules of the protein L domain, related by a noncrystallographic 2-fold axis, as revealed by a self-rotation function calculated with native diffraction data. © 1995 Wiley-Liss, Inc.     

Factors influencing first-year medical students' perceptions of stress

This preliminary report from an ongoing longitudinal study of students at both Tufts and Harvard medical schools examined the relationship between premedical school demographic and stress variables and the numbers and types of stresses reported by students in open-ended interviews conducted during their first year of medical school. Analyses showed that the general categories of "medical school stresses" and "social stresses related to medical school" accounted for about two-thirds of the total number of stresses mentioned. The overall distributions of stresses across general categories were remarkably similar in all student groups studied, although significant differences were found between students from different background groups on a number of the specific types of stress. No significant differences were found between groups as to those reporting a high total number of stresses. There did appear to be differences in the degree to which students felt affected by the stresses they reported, suggesting the need for additional data on the meanings of different stresses for different students and the ways in which they cope with them.     

Effects of intravenous infusions of prostaglandin D2 in man

Prostaglandin D₂ (PGD₂) was infused intravenously into normal male volunteers. Seven subjects received infusions of 16, 32, 64 ng/kg/min and six of these a further dose of 128 ng/kg/min. Each individual's maximum dose was limited by discomfort caused by intense facial flushing and nasal congestion. At these doses there was no significant effect on systolic or diastolic blood pressure nor on spirometric measurements. There was a small but statistically significant tachycardia at 64 and 128 ng/kg/min. Collagen- and adenosine diphosphate (ADP)-induced platele aggregation was not affected at any of the infusion rates. Infused PGD₂ is unlikely to be a useful antithrombotic agent.     

Metalloproteinase inhibitors from bovine cartilage and body fluids

Inhibitors of the mammalian metalloproteinases, collagenase, proteoglycanase and gelatinase were isolated from bovine cartilage (extracts and culture medium) and bovine amniotic fluid and serum. These inhibitors either bind or do not bind to concanavalin-A--Sepharose, with Mr (gel filtration) of about 30 000 and 20 000, respectively. Cartilage and chondrocyte culture media contained only concanavalin-A-binding inhibitors whereas cartilage extracts contained only a non-binding inhibitor: serum and amniotic fluid contained both forms of inhibitory activities. In moist biochemical respects, particularly in their abilities to inhibit metalloproteinases, all of the inhibitors were found to be similar. It is concluded that the forms of the inhibitors that differ in Mr may be closely related to the tissue inhibitor of metalloproteinases (TIMP) previously purified from rabbit and human sources. These findings help to clarify other studies on collagenase inhibitors and support the concept that TIMP-like inhibitors may be important in the control of connective tissue degradation.     

Photoinactivation of Detergent-Solubilized Plasma Membrane ATPase from Rosa damascena: Action Spectra

The photochemistry of vesicular and detergent-solubilized preparations of plasma membrane-associated ATPase was investigated in Rosa damascena. The cholate-solubilized ATPase activity fractionated into two peaks on a Sephadex G-150 column with simple, but different ultraviolet (UV) sensitivities. The larger enzyme was UV sensitive; the smaller enzyme was relatively insensitive. The activity of both ATPase fractions depended on environment: both were inactive in cholate, relatively inactive in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and active in phosphatidylglycerol and phosphatidylserine. The UV sensitivities of both fractions also depended on their environment. For the UV sensitive fraction, the action spectrum differed in the 300 to 400 nanometers range when the fraction was irradiated with and without lipids. For the resistant fraction, UV sensitivity at 290 nanometers differed (up to 6-fold) in different lipids. The resistant fraction solubilized in octylglucoside had an action spectrum very different from that in cholate or in lipid vesicles. The absorption spectra of the different preparations reflected the action spectra. For both UV sensitive and insensitive fractions, the action spectra for photoinactivation had peaks at 290 nanometers, suggesting that the chromophores were tryptophanyl residues. The loss of ATPase activity was strictly correlated with the loss of fluorescence from tryptophan in the partially purified enzymes. Cs⁺ protected the UV sensitive activity but not the insensitive one. We propose a model which explains the difference in UV sensitivities based on the positions of the tryptophan residues in the two proteins.