Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell.

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.

A novel mechanism by which silica defends grasses against herbivory.

BACKGROUND AND AIMS: Previous studies have shown that silica in grass leaves defends them against small herbivores, which avoid high-silica grasses and digest them less efficiently. This study tested the idea that silica can reduce digestibility by preventing the mechanical breakdown of chlorenchyma cells. METHODS: Both the percentage of total chlorophyll liberated from high- and low-silica grass leaves by mechanical grinding and the chlorophyll content of locust faeces were measured. KEY RESULTS: High-silica grasses released less chlorophyll after grinding and retained more after passing through the gut of locusts, showing that silica levels correlated with increased mechanical protection. CONCLUSIONS: These results suggest that silica may defend grasses at least in part by reducing mechanical breakdown of the leaf, and that mechanical protection of resources in chlorenchyma cells is a novel and potentially important mechanism by which silica protects grasses.

Association of the major coat protein of fd bacteriophage with phospholipid vesicles.

The association of the major coat protein of fd bacteriophage with a phospholipid bilayer was investigated by analyzing the protein's susceptibility to proteolysis and its circular dichroism spectrum when incorporated into single-walled phospholipid vesicles. In the limits tested, this association appeared to be independent of the mass ratio of protein to lipid and of vesicle size, phospholipid composition, and method of preparation. The circular dichroism data are consistent with a similar "membrane-bound" conformation for all cases of vesicle-associated coat protein and for deoxycholate micelle-associated coat protein. Proteolysis of coat protein associated with deoxycholate micelles and with phospholipid vesicles defined the central hydrophobic core presumed to represent that portion of the protein which associates with membrane bilayers in vivo. The isolated core, which assumed a predominantly beta-type conformation in detergent solution, maintained a beta conformation when associated with a vesicle phospholipid bilayer.

The pI and pXI assembly proteins serve separate and essential roles in filamentous phage assembly.

Three non-capsid, phage-encoded proteins, pI, pIV and pXI, are required for assembly of the filamentous bacteriophage at the envelope of Escherichia coli. pIV forms the outer membrane component of the assembly site, and pI and pXI are predicted to form the cytoplasmic membrane component. pXI is the result of an in-frame internal translational initiation event in gene I and is identical with the carboxyl-terminal third of pI in amino acid sequence, membrane localization and topology. The two proteins share a cytoplasmic domain predicted to be an amphipathic helix, a transmembrane domain, and a periplasmic domain. By mutating the initiation site for pXI, a phage was made that produced only pI and was shown to absolutely require functional plasmid-encoded pXI for growth. Further mutational analysis was done to examine the functional determinants of the amphipathic helix and periplasmic domains of the pI and pXI proteins. The results show that the amphipathic helix region is very important for pI function but not for pXI function. Mutational analysis of the periplasmic domains of pI and pXI implies that these domains also perform separate functions, and suggests that the interaction between pI and pIV in the periplasm is critical for assembly. The results are discussed with regard to the separate roles that the pI and pXI proteins play in the overall process of phage assembly.

The major coat protein of filamentous bacteriophage f1 specifically pairs in the bacterial cytoplasmic membrane.

Filamentous bacteriophage are long, thin single-stranded DNA viruses that infect male strains of Escherichia coli without killing the host. Each phage contains approximately 2700 copies of the major coat protein, pVIII, which exists as a 5.2 kDa cytoplasmic membrane protein prior to incorporation into phage. Studies from a number of groups analyzing the behavior of wild-type and mutant pVIII in detergents suggested that pVIII might pair under these conditions. In order to test whether pVIII molecules pair in vivo in the cytoplasmic membrane, four plasmidencoded pVIII variants were constructed in which specific residues in the transmembrane region were mutated to cysteine in an attempt to stabilize any pair via disulfide bridges. Variants A35C and I39C were unable to complement phage with an amber mutation in gene VIII. The I39C variant was unable to be packaged into phage particles even though it was inserted into the membrane. In the case of A35C, the inability to complement was not due to a packaging defect because the variant protein could be packaged into phage in the presence of wild-type pVIII. Western blot analysis of cytoplasmic membrane samples revealed that the A35C variant formed stable disulfide dimers in vivo. Expression of A35C interfered with wild-type phage infection, indicating that the assembly machinery may recognize the disulfide dimers in some non-productive way. The results indicate that pVIII may specifically pair along a particular face in the cytoplasmic membrane prior to assembly; however, these pairs must be able to be separated in order for normal assembly to occur.

The products of gene I and the overlapping in-frame gene XI are required for filamentous phage assembly.

The class I filamentous bacteriophage are non-lytic single-stranded DNA phage, which are assembled at the cell envelope as they are extruded from the Gram-negative bacteria, Escherichia coli. The process requires the products of the phage genes I and IV, which reside in the inner and outer membrane, respectively, and are not present in the mature phage particle. Gene I encodes two proteins, the full length 348-residue pI and a smaller pI*, which this report shows is the result of an internal translation initiation event at methionine codon 241. Both pI and pI* are shown to be required for phage assembly. Therefore, pI* can be considered the product of an additional phage gene, XI, which is a separate in-frame gene that overlaps gene I. Both proteins contain a 13-residue region adjacent to the cytoplasmic face of the inner membrane that probably exists as a positively charged amphiphilic helix. Although this region is not required for membrane insertion of pI and pI*, it is shown to be required for phage assembly. Oligonucleotide-directed mutagenesis of this region, which removes positive charges or alters the hydrophobic face of the putative helix, renders pI and pI* unable to function in phage assembly. This region of pI and pI* is highly homologous in structure to the carboxyl-terminal 11 amino acids of pVIII, the main coat protein, which also reside adjacent to the cytoplasmic face of the inner membrane.

Morphogenesis of f1 filamentous bacteriophage. Increased expression of gene I inhibits bacterial growth.

We have cloned the gene I sequence of the filamentous bacteriophage f1 downstream from the lambda leftward promoter on a plasmid that also contains the temperature-sensitive lambda repressor, cI857. Temperature induction of gene I protein (pI) resulted in rapid cessation of growth. This inhibition appears to involve a rapid decrease in synthesis of host protein and RNA. The ability of pI to cause this inhibition is not dependent on thioredoxin, a host factor that is necessary for phage morphogenesis and has been shown by genetic data to interact with pI. The inhibition does not appear to be mediated by the amino half of the protein, as induction of an identical plasmid construction of an amber mutant positioned two-thirds along gene I, does not affect cell growth. Analysis of the transcription products from the cloned gene I confirmed previous suggestions that a transcription terminator exists in the amino-terminal portion of the gene. In addition, there is no detectable promoter activity in the 152 bases immediately upstream from the gene. These data and the inability to overproduce pI argue for down-regulation of pI production. Radioactive labeling of proteins in maxi-cells and normal Escherichia coli cells identifies pI as a protein of about 39,000 Mr that partitions with the cell envelope. Pulse-chase experiments suggest that pI is not processed to any appreciable extent.

Nucleotide sequence of the F plasmid gene, traC, and identification of its product.

The traC gene of the F plasmid tra operon is required for the assembly of mature F-pilin subunits into extended F pili. The nucleotide sequence of traC was determined with a determined with a deduced coding region of 875 amino acids (aa) and 99066 Da. The traC1044 mutant allele, which allows filamentous phage infection in the absence of piliation, contains a C-to-T transition leading to an Arg----Cys substitution. Confirmation of the translational start came from the direct N-terminal aa sequencing of a TraC-alkaline phosphatase fusion protein.

Temporal stability of the Learning Efficiency Test-II for adults.

This study examined the temporal stability of the Learning Efficiency Test-II with 101 undergraduate students over a mean test-retest time interval of 19 days. Temporal stability estimates were .79 for the Visual Modality factor score, .75 for the Auditory Modality factor score, and .80 for the Global Memory factor score. A repeated measures analysis of variance for these three factor scores indicated no significant mean differences from Test 1 to Test 2. Slightly lower retest correlations were obtained for each of the 12 subtests, with correlations ranging from .44 to .70. The findings indicate that some subtests are reliable to assess the memory processing of adults over time and also highlight the stability over 19 days of memory processes for intact learners. Other studies with older groups of persons are needed to examine the stability of scores.

Leisure: how to promote inpatient motivation after discharge.

1. A group of psychiatric inpatients followed plans to participate in leisure activities after discharge at significantly higher levels when treated with classes and written contracts; or classes, written contracts, and musical entertainment; rather than with classes alone. 2. Inpatient classes for psychiatric patients should be coupled with other interventions, such as contracts or musical entertainment, to assist patients to attain therapeutic goals after discharge. 3. The utilization of written goal-setting contracts seems to have merit as a routine nursing intervention with psychiatric inpatients. 4. The use of a written contract left with the patient may promote or enhance a nurse/patient transaction.

Utility of the WAIS in predicting vocational success of psychiatric patients.

Examined the utility of the WAIS OA, BD, PIQ and FSIQ scores in combination with age, apparent degree of emotional severity and psychiatric disability in predicting the vocational success of 180 psychiatric outpatients. Although no significant statistical differences were obtained in a cros-validation procedure indicating that psychiatric disability affected the efficacy of the derived regression equations, substantial differences in predictable variance accounted for were found to be related to the nature of the psychiatric disability. This must be attended to in attempts to predict the vocational success of psychiatric outpatients.

The tol gene products and the import of macromolecules into Escherichia coli.

Genetic studies have identified a number of genes whose products appear to be required for the transport of the group A colicins and the single-stranded DNA of certain filamentous bacteriophages into Escherichia coli. Mutations in these genes allow normal binding of the colicins to their outer-membrane receptors and of the bacteriophage of the tip of specific conjugative pili, but do not allow translocation of the macromolecules to their target. These mutations have been designed 'tolerant' (tol) mutations and the protein products specified by these genes appear to comprise part of a transport system known as the Tol import system. Some of these genes have been isolated, sequenced and their protein products localized to the membranes or periplasm of E. coli. Information is also available regarding the domains of the colicins or phage proteins which interact with the Tol proteins. A preliminary model of the location and possible interactions of the Tol proteins is presented.

An amino acid sequence which directs membrane insertion causes loss of membrane potential.

A 55-amino acid segment, normally present between residues 241 and 295 of the 348-residue gene I protein of the filamentous bacteriophage f1, acts as an internal signal sequence for gene I protein or, when present in fusion proteins, for EcoRI endonuclease or alkaline phosphatase. The resulting proteins are inserted so that they span the membrane with sequences on the amino side of the 55-residue segment in the cytoplasm and those near the carboxy side outside the cytoplasmic membrane. The presence of these proteins in the membrane results in the rapid inhibition of cell growth, probably from a loss of the membrane potential. We describe some of the elements in this 55-residue segment that appear to be crucial for its interaction with the membrane.