Evaluation of laser-Doppler perfusion imaging for measurement of blood flow in cortical bone.

Most techniques currently available to measure blood flow in bone are time consuming and require destruction of the tissue, but laser-Doppler technology offers a less invasive method. This study assessed the utility of laser-Doppler perfusion imaging (LDI) to measure perfusion in cortical bone. Twelve mature New Zealand White rabbits were assigned to one of three groups: normal control, constriction (norepinephrine), or dilatation (nitroprusside). The left and right medial tibiae were consecutively scanned at red (634-nm) and near-infrared (810-nm) wavelengths to examine the repeatability of LDI output. The pharmacological intervention groups were injected with the respective drug, and LDI measurements at 810 nm were obtained concurrently with colored microsphere-determined flow in all of the groups. LDI effectively quantified blood flow in cortical bone and detected physiologically induced changes in perfusion. A significant positive correlation was found between microsphere-determined flow and LDI output (r = 0.6, P < 0.05). Repeatability of consecutive LDI measurements was within 5%. The effectiveness of LDI to measure perfusion in bone suggests this method has potential for investigating the role of blood flow in bone metabolism and remodeling.     

Identification of a male-specific (H-Y) antigen on the flagellar plasma membrane of ram epididymal spermatozoa

Summary H-Y (male-specific) antigen has been detected on the plasma membranes of both caput and caudal ram spermatozoa using both immunoperoxidase and immunofluorescence labelling techniques. In these spermatozoa the distribution of H-Y antigen appears to be confined to both the posterior region of the head and the mid-piece region of the flagellum. In addition, caput spermatozoa also exhibit intense immunoperoxidase staining of the cytoplasmic droplet which is situated on the flagellum at the base of the head. Western blot analyses of purified plasma membranes from the flagella of caudal spermatozoa have revealed the presence of a malespecific protein with an estimated molecular weight of 25,000–27,000.     

Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

Characterization and sequence analyses of antibody-selected antigenic variants of herpes simplex virus show a conformationally complex epitope on glycoprotein H.

Thirteen antigenic variants of herpes simplex virus which were resistant to neutralization by monoclonal antibody 52S or LP11 were isolated and characterized. The antibodies in the absence of complement potently neutralize infectivity of wild-type virus as well as inhibit the transfer of virus from infected to uninfected cells ("plaque inhibition") and decrease virus-induced cell fusion by syncytial strains. The first variant isolated arose in vivo. Of 66 type 1 isolates analyzed from typing studies of 100 clinical isolates, one was identified as resistant to neutralization by LP11 antibody. The glycoprotein H (gH) sequence was derived and compared with those of wild-type and syncytial laboratory strains SC16, strain 17, and HFEM. The sequences were highly conserved in contrast to the diversity observed between gH sequences from herpesviruses of different subgroups. Only four coding changes were present in any of the comparisons, and only one unique coding change was observed between the laboratory strains and the clinical isolate (Asp-168 to Gly). These sequences were compared with those of antigenic variants selected by antibody in tissue culture. Twelve variants were independently selected with antibody LP11 or 52S from parent strain SC16 or HFEM. For each variant, the gH nucleotide sequence was derived and a point mutation was identified giving rise to a single amino acid substitution. The LP11-resistant viruses encoded gH sequences with amino acid substitutions at sites distributed over one-half of the gH external domain, Glu-86, Asp-168, or Arg-329, while the 52S-resistant mutant viruses had substitutions at adjacent positions Ser-536 and Ala-537. One LP11 mutant virus had a point mutation in the gH gene that was identical to that of the clinical isolate, giving rise to a substitution of Asp-168 with Gly. Both LP11 and 52S appeared to recognize distinct gH epitopes as mutant virus resistant to neutralization and immunoprecipitation with LP11 remained sensitive to 52S and the converse was shown for the 52S-resistant mutant virus. This is consistent with previous studies which showed that while the 52S epitope could be formed in the absence of other virus products, virus gene expression was required for stable presentation of the LP11 epitope, and for transport of gH to the cell surface (Gompels and Minson, J. Virol. 63:4744-4755, 1989). All mutant viruses produced numbers of infectious particles that were similar to those produced by the wild-type virus, with the exception of one variant which produced lower yields.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cloning of the Escherichia coli release factor 2 gene.

The protein release factor 2 (RF2) participates in Escherichia coli polypeptide chain termination with codon specificity (UAA or UGA). A colicin E1 recombinant identified in the Carbon and Clarke E. coli bank contains the protein release factor 2 gene. A 1.7-kilobase E. coli fragment has been subcloned into the plasmid pUC9 vector. Bacterial cells, containing the plasmid recombinant, produce elevated levels of protein release factor 2 as detected by an immune precipitation assay and in vitro measurement of UGA-directed peptide chain termination and [3H]UGA codon recognition.     

Chemokinetic accumulation of human neutrophils on immune complex-coated substrata: analysis at a boundary

The locomotory behavior of human blood neutrophil leukocytes was studied at a boundary between two surfaces with different chemokinetic properties. This was achieved by time-lapse cinematography of neutrophils moving on coverslips coated with BSA, then part-coated with immune complexes by adding anti-BSA IgG with a straight-line boundary between the BSA and the immune complexes. Cell locomotion was filmed in microscopic fields bisected by the boundary, and kinetic behavior was assessed by comparing speed (orthokinesis), turning behavior (klinokinesis), and the rate of diffusion of the cells on each side of the boundary, using a recently described mathematical analysis of kinesis. In the absence of serum or complement, the proportion of motile cells and their speed and rate of diffusion were greater on BSA than on antiBSA, but there was no consistent difference in turning behavior between cells on the two surfaces. The immune complexes were therefore negatively chemokinetic in comparison with BSA, and this resulted from a negative orthokinesis with little or no contribution from klinokinesis. As would be predicted theoretically, this resulted in gradual accumulation of cells on the immune complexes even in the absence of a chemotactic factor. In further studies, a parallel plate flow chamber was used to show that, under conditions of flow, neutrophils accumulated much more rapidly on a surface coated with BSA- anti-BSA than on BSA alone. Moreover, neutrophils on immune complex- coated surfaces lost their ability to form rosettes with IgG-coated erythrocytes. This suggests that neutrophils on immune complex-coated surfaces redistribute their Fc receptors (RFc gamma) to the under surface, and that the lowered speed of locomotion is due to tethering of neutrophils by substratum-bound IgG-Fc.     

Characteristics and N-terminal amino acid sequence of a manganese peroxidase purified from Lentinula edodes cultures grown on a commercial wood substrate

Summary Extracellular culture filtrates from ligninolytic cultures of the lignin-degrading basidiomycete Lentinula (syn. Lentinus) edodes (Berk.) Pegler contained one major peroxidase when grown on a commercial oak-wood substrate. The peroxidase was purified by polyethylenimine clarification, anion-exchange chromatography, and hydrophobic-interaction HPLC. The enzyme (MnP1) was a heme-iron protein with an apparent molecular weight of 44 600 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and an isoelectric point of pH 3.2. The native enzyme had an absorption maximum at 407 nm, which shifted to 420 nm upon H₂O₂ addition. The pyridine-hemochrome-absorption spectrum indicated that one heme group was present per enzyme as protoporphyrin IX. N-terminal amino acid sequencing showed that MnP1 had higher sequence homology with manganese peroxidases than with lignin peroxidases reported from Phanerochaete chrysosporium. L. edodes MnP1 was capable of oxidizing lignin and lignin-model compounds in the presence of manganese and H₂O₂.On leave from the Department of Biochemistry, University of Otago, P. O. Box 56, Dunedin, New Zealand.Research carried out while a visiting scientist at the USDA Forest Products Laboratory from the National Chemistry Laboratory, Pune, India 41 1008 Offprint requests to: I. T. Forrester