Human bloodstains: individualization by crossed electroimmunodiffusion.

Crossed electroimmunodiffusion was evaluated as a means for establishing the individuality ("fingerprinting") of human bloodstains. In ten separate examinations on stains from each of ten persons there was at least one peak with a unique range in height so that individualization was possible. The heights of certain peaks showed statistically significant female-male differences.

Spring shoot growth in douglas-fir may be initiated by gibberellins exported from the roots.

Trials conducted under controlled environments demonstrated that the delay of bud activity of Douglas-fir (Pseudotsuga menziesii) seedlings occasioned by low temperature of the soil could be eliminated by application of gibberellic acid. Analyses of field-grown plants showed a parallel increase in bud activity, level of gibberellin-like compounds in xylem sap, and soil temperature during February and March.

Evolution of permease diversity and energy-coupling mechanisms with special reference to the bacterial phosphotransferase system.

Different classes of apparently unrelated permeases couple different forms of energy to solute transport. While the energy coupling mechanisms utilized by the different permease classes are clearly distinct, it is proposed, based on structural comparisons, that many of these permeases possess transmembrane, hydrophobic domains which are evolutionarily related. Carriers may have arisen from transmembrane pore-forming proteins, and the protein constituents or domains which are specifically responsible for energy coupling may have had distinct origins. Thus, complex permeases may possess mosaic structures. This suggestion is substantiated by recent findings regarding the evolutionary origins of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). Mechanistic implications of this proposal are presented.

Cloning, mapping and gene product identification of rhaT from Escherichia coli K12.

Rhamnose utilization requires the function of a specific rhamnose transport system. Rhamnose transport mutants have been isolated and characterized. The structural gene, rhaT, encoding the rhamnose permease has been cloned from Escherichia coli. rhaT has been mapped in the rha locus (87.7 min) by analysis of cotransduction with glpK and other rha markers. The precise location of the gene has been determined by complementation analysis of rhamnose transport mutants transformed with recombinant plasmids containing different fragments of the cloned region. Gene order (counterclockwise) is established as glpK . . . rhaT-rhaR-rhaS-rhaB-rhaA-rhaD. The gene product has been identified by expression of rhaT in a T7 RNA polymerase/promoter system. This 23 kDa protein has been assigned to the rhaT product and has been shown to be located in the cell membrane.

Studies by crossed electroimmunodiffusion on the individuality and sexual orgin of bloodstains.

Crossed electroimmunodiffusion (CEID) was evaluated as a means for individualizing human blood stains by studying variations within and among individuals in 22 serum antigens in ten subjects over a four-month period. The extent of variation within an individual was determined by making CEID runs on blood stains obtained on ten different occasions and measuring the precipitin peak heights produced by each of the 22 antigens. When the range in height of any particular peak was completely different in subject-subject comparisons, the peak was judged to be of value in individualization. By this criterion, each of the ten subjects could be distinguished from all others (65 subject-subject distinctions), but in most cases the distinction had to be based on difference in 5 or less of the 22 antigens. The antigens of value in distinguishing among males were largely different from those of value in distinguishing among females. Overall, the antigens of greatest value in individualization were 8, 9, 10, and 11. Only one of these (10, ceruloplasmin) could be identified as a particular serum proteins.

NFP and ecological mothering.

PIP: John F. Kippley's Couple to Couple League, in addition to providing instruction in the natural family planning (NFP) method, conducts a series of 4 classes for couples who want to learn about health care, breastfeeding, prepared childbirth, and continence. The parents learn the philosophy and practice of the "ecology of natural mothering". Like prepared childbirth, NFP is based on scientific principles and is an art which must be mastered through study and practice. "Ecological mothering" means total breastfeeding and physical closeness between mother and infant. The basic principle is that the baby's frequent sucking will provide the mother with a period of natural infertility. The concept of total breastfeeding is radically different from the cultural nursing as is often seen in this society. "Ecological mothering" means: no solid foods for the 1st 6 months of the baby's life, no artificial pacifiers or nipples, no schedules, and mother baby closeness day and night. The vast majority of women will have extended natural infertility with ecological breastfeeding, and there will be a menstrual period after childbirth before ovulation occurs again. After the 1st menstrual period postpartum, a couple should assume that fertility has returned. Then it is time for them to use their awareness of a woman's body signals to determine fertility. The Couple to Couple League maintains that a woman can "read" her body through the symptothermal changes that are observed and then recorded. Actually, interpreting these bodily changes accurately allows a couple to detect ovulation before the 1st menstrual period postpartum. A chart outlines the theory of the symptothermal system. The observations of body changes can help a couple either to planor avoid conception. The symptoms and temperature changes are charted and intercourse is avoided during peak fertility if the couple wants to avoid conception. Kippley notes that the philosophy behind NFP has brought many couples closer together.^ieng

Seed orchards in development.

This paper reviews the role of seed orchards as output systems for genetic improvement programs. In this paper, technological changes since the 1950s are examined, with emphasis on recent developments. The need to equate the type of seed orchard to the type of forestry practiced is recognized, as is the significance of the relationship between seed orchards and other output systems for genetically improved material.

A diffusible auxin from Pinus radiata pollen and its possible role in stimulating ovule development.

Germinated pollen of Pinus radiata contains an auxin which is active in the Avena coleoptile test. It differs from all other hormones detected in pine pollen in that it is readily able to diffuse out from the pollen into an agar medium. It is suggested that, following pollination in vivo, the auxin may diffuse from the germinated pollen-tube onto the nucellus, thereby triggering the processes which allow ovule and gametophyte development to proceed. The auxin is water-soluble and may be an indole derivative.

Evaluation of fluorescein isothiocyanate-labeled whole antiserum in the immunofluorescent identification of microorganisms.

Portions of a whole antiserum to Histoplasma capsulatum were reacted with amounts of fluorescein isothiocyanate (FITC) that ranged from 50 to 400 mug/mg of protein. Portions of the globulin from the same antiserum were reacted with amounts of FITC that ranged from 12.5 to 50 mug of FITC per mg of protein. The globulin conjugates (postlabeled globulins), the whole serum conjugates, and the globulins from the whole serum conjugates (prelabeled globulins) were compared with respect to their fluorescein-protein (F:P) ratios and fluorescent-antibody (FA) activities. The whole serum sample treated with 50 mug of FITC per mg of protein was least reactive in FA tests, and its globulin had the lowest F:P. All other conjugates had globulins with F:P ratios that were considered to be adequate for high FA activity. It was found, however, that the prelabeled globulins were considerably less reactive than the postlabeled globulins or the whole serum conjugates. A larger amount of brightly staining reagent per milliliter of original serum could be obtained from labeled whole serum than from postlabeled globulin. Lissamine-rhodamine conjugated to bovine serum albumin (LRBSA) was evaluated as a counterstain to be used in conjunction with FITC-labeled whole antisera. The counterstain was effective in masking nonspecific FITC fluorescence in Formalin-fixed tissues and in culture smears of fungi. Masking was incomplete in culture smears of a bacterium and in blood smears containing a protozoan.

Experimental histoplasmosis in the mouse. Immunoglobulin class response, total immunoglobulin A levels, immune complex formation, and occurrence of antigen in serum and urine.

Biotin-avidin enzyme-linked immunosorbent assays (ELISA) were developed to study the appearance of free antibody of the IgM, G, and A classes of immune complexes (IC) containing those classes and of antigen in the serum and urine of mice with experimentally induced, disseminated histoplasmosis (histo) over a period ranging from 4 to 64 days after infection. Free IgM was detected 4 days after infection, remained at low levels, and disappeared on Day 64, whereas free IgG was first detected on Day 7 and rose to very high levels before declining on Day 64. Free IgA was detected only once, on Day 21. IC containing IgM were seen first on Day 4, remained at low levels, and became undetectable on Day 64. IC containing IgM were detected on Day 7, peaked on Day 14, and declined through Day 64. IC containing IgA wee seen at low levels on Days 7, 14, and 21. Estimation of total IgA levels, done by single radial immunodiffusion, showed a statistically significant decrease on Day 14, followed by a slightly significant increase on Days 21 and 30. Antigen was detected in as much as 80% of serum specimens and 100% of urine samples during the first 2 wk postinfection but rarely afterwards. We conclude that ELISA provides a highly sensitive way to study antibody, IC, and antigen in host body fluids during histo infection and that IgM and antigen detection can be very early indices of infection. Measurements of IC and total IgA seem to be of relatively less importance in detection of infection.

Molecular analysis of the glpFKX regions of Escherichia coli and Shigella flexneri.

We have identified a new gene, glpX, belonging to the glp regulon of Escherichia coli, located directly downstream of the glpK gene. The transcription of glpX is inducible with glycerol and sn-glycerol-3-phosphate and is constitutive in a glpR mutant. glpX is the third gene in the glpFKX operon. The function of GlpX remains unknown. GlpX has an apparent molecular weight of 40,000 on sodium dodecyl sulfate-polyacrylamide gels. In addition to determining the E. coli glpX sequence, we also sequenced the corresponding glpFKX region originating from Shigella flexneri, which after transfer into E. coli was instrumental in elucidating the function of glpF in glycerol transport (D. P. Richey and E. C. C. Lin, J. Bacteriol. 112:784-790, 1972). Sequencing of the glpFKX region of this hybrid strain revealed an amber mutation instead of the tryptophan 215 codon in glpF. The most striking difference between the E. coli and S. flexneri DNA was found directly behind glpK, where two repetitive (REP) sequences were present in S. flexneri, but not in the E. coli sequence. The presence or absence of these REP sequences had no effect on transport or on growth on glycerol. Not including the REP sequence-containing region, only 1.1% of a total of 2,167 bp sequenced was different in the two sequences. Comparison of the sequence with those in the EMBL data library revealed a 99% identity between the last third of glpX and the first part of a gene called mvrA. We show that the cloned mvrA gene (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988) originated from the 88-min region of the Escherichia coli chromosome and not, as reported, from the 7-min region and that the gene product identified as MvrA is in fact encoded by a gene distal to glpX.

Glycerol kinase of Escherichia coli is activated by interaction with the glycerol facilitator.

Glycerol transport is commonly cited as the only example of facilitated diffusion across the Escherichia coli cytoplasmic membrane. Two proteins, the glycerol facilitator and glycerol kinase, are involved in the entry of external glycerol into cellular metabolism. The glycerol facilitator is thought to act as a carrier or to form a selective pore in the cytoplasmic membrane, whereas the kinase traps the glycerol inside the cell as sn-glycerol-3-phosphate. We found that the kinetics of glycerol uptake in a facilitator-minus strain are significantly different from the kinetics of glycerol uptake in the wild type. Free glycerol was not observed inside wild-type cells transporting glycerol, and diffusion of glycerol across the cytoplasmic membrane was not the rate-limiting step for phosphorylation in facilitator-minus mutants. Therefore, the kinetics of glycerol phosphorylation are different, depending on the presence or absence of the facilitator protein. We conclude that there is an interaction between the glycerol facilitator protein and glycerol kinase that stimulates kinase activity, analogous to the hexokinase- and glycerol kinase-porin interactions in mitochondria.

Flurorcitrate resistant tricarboxylate transport mutants of Salmonella typhimurium.

Spontaneous and Tn10 induced fluorocitrate resistant mutants were isolated and characterized. These mutants were unable to grow on either cis-aconitate or DL-isocitrate but were still able to grow slowly on sodium citrate and normally on potassium or potassium-plus-sodium citrate. These mutants were defective in both citrate transport and citrate binding to priplasmic proteins. Tn10 insertion mutants were unable to produce immunologically detectable amounts of the citrate inducible periplasmic C protein previously shown to bind tricarboxylates. Using a series of tct::Tn10 directed Hfrs the tct locus was accurately positioned at 59 units between srlA and pheA, but was not cotransducible with either gene. In the absence of P22 mediated cotransduction with 16 adjacent chromosomal markers the srlA and tct loci were bridged by using a series of tct flanking Tn10 insertions, and by newly isolated and characterized nalB mutants. In addition the hyd and recA loci were located establishing the gene order in this region of the chromosome as: pheA tct nalB recA srlA hyd cys. Nitrosoguanidine derived tricarboxylate mutations (Imai 1975) were also mapped within the tct locus.

Physical and genetic structure of the glpD-malT interval of the Escherichia coli K-12 chromosome. Identification of two new structural genes of the glp-regulon.

A transducing lambda phage carrying glpD''lacZ, glpR, and malT was isolated from a strain harboring a glpD''lacZ fusion. Comparison of restriction endonuclease cleavage patterns of DNA isolated from this phage with that of the previously cloned malT region (Raibaud and Schwartz 1980) facilitated the construction of recombinant plasmids carrying different portions of the glpD-malT region. Results of minicell analysis and complementation studies showed that this region of the chromosome encodes at least five polypeptides. These included the previously identified glpD, glpR, and malT gene products. In addition, two new structural genes of the glp regulon (glpE and glpG) located between the glpD and glpR genes were identified. Hybrid plasmids carrying glpD''lacZ and glpR''lacZ fusions were constructed. Restriction endonuclease cleavage analysis of these two plasmids demonstrated that glpD and glpR are divergently transcribed.

Immunochemical studies on burro antibodies to Histoplasma capsulatum.

Burro antiserum to Histoplasma capsulatum was studied from the viewpoint of precipitating antibody distribution among euglobulins, pseudoglobulins, and immunoglobulin classes. By immunodiffusion analysis it was determined that throughout immunization most of the antibody was euglobular, but there was an increase in pseudoglobular antibody as immunization progressed. By immunoelectrophoretic and immunodiffusion analyses of specifically purified antibody, and antibody non-specifically fractionated by column chromatography and ultracentrifugation, it was established that there was no demonstrable IgM antibody, that most of the antibody appeared to be IgG, and that a gamma1 immunoglobulin, probably IgA or T-globulin, may have been responsible for some of the activity. No major changes in the distribution of antibody among immunoglobulin classes seemed to occur during immunization.

Purification and properties of a citrate-binding transport component, the C protein of Salmonella typhimurium.

Salmonella typhimurium was shown to contain a citrate-binding protein (C protein) which was purified to homogeneity from the periplasmic fraction released by cold osmotic shock. The protein is dimeric, has an apparent molecular weight of 28 000 and an isoelectric point of 6.1. Sodium ions were required for optimum substrate binding, however, the divalent cations Zn2+, Mg2+, and Co2+ were inhibitory. The C protein was relatively stable but sensitive to various detergents and chaotropic agents. Approximately one citrate molecule was bound per molecule of protein and citrate binding (Kd = 1-2.6 microM) was strongly competitively inhibited by DL-isocitrate and DL-fluorocitrate but not by other carboxylates. Neither succinate, glutamate, nor acetate were bound to the C protein. No apparent enzyme activity was associated with this protein. A concomitant reduction in the level of binding protein and in citrate transport activity occurred in osmotically shocked cells as well as with L-malate- or succinate-grown cells. Fluorocitrate-resistant mutants were simultaneously defective in citrate transport, citrate binding, and production of cross-reacting material. One transport-defective mutant did produce citrate binding protein.

Antigens of human cytomegalovirus: electroimmunodiffusion assay and comparison among strains.

The antigens of strain AD169 of human cytomegalovirus (CMV) were extracted by various methods and at different times following the appearance of cytopathic effects (c.p.e.) in infected fibroblasts. Assay with a pooled human serum in electroimmunodiffusion (EID) revealed that the most reactive preparations were obtained by shell-freeze (SF) extraction on the fourth day after 4+c.p.e. As many as 20 antigens could be detected in the original gels, most of which were stable upon storage at 4 degrees C for up to 4 weeks; of these, about 14 can be reproducibly seen on photographs. EID runs on day 4 SF preparations from high-passage CMV strains C87 and Davis and low passage recent isolates VD14, 1694 and 1723 resolved, respectively, 15, 15, 13, 11 and 11 antigens in the original gels (11, 9, 11,8 and 9 are visible in photographs). Strains 1694 and 1723 shared fewer antigens with one another and with high passage strains than were shared among the latter, whereas VD14 had relatively large numbers of antigens common to both low and high passage strains. At least six antigens were common to all strains.

Detection of antibodies to cytomegalovirus by an improved counterimmunoelectrophoretic procedure.

An improved counterimmunoelectrophoretic (CIE) procedure for detection of antibodies to human cytomegalovirus (CMV) is described. The procedure, which uses high ionic strength buffer and concentrated antigen, is as sensitive and specific as complement fixation (CF), indirect hemagglutination (IHA), and anti-complement immunofluorescence (ACIF) but requires concentration of some sera. In studies on sera from 118 normal blood donors, the percent correlation of CIE results with those by CF, IHA, and ACIF were 97.5, 98.3, and 98.3, respectively, and exceeded slightly the correlations among the latter three procedures. The appearance of precipitation immediately or early after a CIE run and of more than one line of precipitate was indicative of high titers by the other procedures, while single lines of late precipitation occurred with most low titer sera but also with many of those of high titer.

Tricarboxylate-binding proteins of Salmonella typhimurium. Purification, crystallization, and physical properties.

Citrate transport in Salmonella typhimurium involves inducible periplasmic components. Two forms of a tricarboxylate-binding protein, C1 and C2, were isolated, in high yield, from the periplasm of a cyclic AMP phosphodiesterase mutant. These immunologically cross-reactive Mr = 29,000 proteins were crystallized using ammonium sulfate. CD measurements indicated considerable secondary structure: 24% a helix, and 12% beta structure. The amino acid compositions of C1 and C2 were identical. The NH2-terminal sequence of C1 was determined; C2 was found to have a blocked NH2 terminus (pyroglutamate). C1 and C2 are products of the same gene (Somers, J. M., and Kay, W. W. (1983) Mol. Gen. Genet. 190, 20-26). C1 and C2 bound a variety of citrate analogues and organic acids, with a predominant specificity for tricarboxylates (citrate KD 1.4 X 10(-7) M), and both required a deprotonated central carboxyl group for binding. Citrate was not bound to C protein as either a salt or metal ion complex.