Lectin-induced deciduoma formation in the pseudopregnant rat

Decidual cell induction in the pseudopregnant rat was examined in this study using the lectin concanavalin A (ConA). The histochemical binding of the lectin to the uterine cell surface at the time of deciduomatic induction was also studied. ConA was found to induce significant deciduomata (decidual-like tissue) in the uterine horn when injected intraluminally on day 5 of pseudopregnancy (PSP). ConA-induced deciduomata appeared as a series of discrete nodules in the uterine horn, reminiscent of the anatomical appearance of normal embryo implantation sites. Deciduoma induction by ConA was greatly reduced by pre-absorption of the lectin with its competitive sugar. Lectin histochemistry revealed binding of ConA to the cell surface on day 5 of PSP. Pre-absorption of the lectin with its competitive sugar also significantly reduced surface binding of the lectin, and this finding may be correlated with the greatly reduced ability of the pre-absorbed lectin to induce deciduomata. Possible mechanisms for the induction of deciduomata by lectins are considered.     

The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization

Summary Conventional solution-phase polymerase chain reaction (PCR) and in situ PCR/PCR in situ hybridization are powerful tools for retrospective analysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archival fixed tissues. Such ailures may not only be due to absent target DNA sequences in the tissues, but may be a direct effect of the type of fixative, fixation time and/or fixation temperature used. The type of nucleic acid extraction procedure applied will also influence amplification results. This is particularly true with in situ PCR/PCR in situ hybridization.To examine these effects in solution-phase PCR, -globin gene was amplified in 100 mg pieces of tonsillar tissue fixed in Formal saline, 10% formalin, neutral buffered formaldehyde, Carnoy's, Bouin's, buffered formaldehyde sublimate, Zenker's, Helly's and glutaraldehyde at 0 to 4°C, room temperature and 37°C fixation temperatures and for fixation periods of 6, 24, 48 and 72 hours and 1 week. DNA extraction procedures used were simple boiling and 5 days' proteinase K digestion at 37°C. Amplified product was visible primarily yet variably from tissue fixed in neutral buffered formaldehyde and Carnoy's, whereas fixation in mercuric chloride-based fixatives produced consistently negative results. Room temperature and 37°C fixation temperature appeared most conducive to yielding amplifiable DNA template. Fixation times of 24 and 48 hours in neutral buffered formaldehyde and Carnoy's again favoured amplification.Fixed SiHa cells (containing 1–2 copies of HPV 16) were examined using PCR in situ hybridization for the amplification of HPV 16. Discrete and diffuse amplification signals were obtained. Neutral buffered formaldehyde fixation for 12–24 hours yielded amplifiable material suitable for use with PCR in situ hybridization. Overall amplification success within cellular preparations was 40%, with non-specific background staining also seen. Possible technical problems encountered with PCR in situ hybridization are discussed.     

HIV-1 integrase blocks infection of bacteria by single-stranded DNA and RNA bacteriophages

Expression of human immunodeficiency virus-1 integrase in Escherichia coli, at levels that had no effect on bacterial cell growth, blocked plaque formation by bacteriophages having single-stranded genomic DNA (M13) or RNA (R17, Q, PRR1). Plaque formation by phages having double-stranded genomic DNA (T4, PR4) was unaffected. Integrase also inhibited infection by the phagemid M13KO7, but it had no effect on production of phage once infection by M13KO7 was established. This result indicated that integrase affects an early stage in infection. Integrase also inhibited phage production following transfection by either single-stranded or double-stranded (replicative form) M13 DNA, it blocked M13 DNA replication, as assayed by incorporation of radioactive nucleotides into DNA, and it failed to affect bacterial pilus function. These data suggest that integrase interacts in vivo with phage nucleic acid, a conclusion supported by studies in which integrase was shown to have a DNA-binding activity in its C-terminal portion. This portion of integrase was both necessary and sufficient for interference of plaque formation by M13 in the present study. Expression of the N-terminal portion of integrase at the same level as intact integrase had little effect on phage growth, indicating that expression of foreign protein in general was not responsible for the inhibitory effect. The simple bacteriophage assay described is potentially useful for identifying integrase mutants that lack single-stranded DNA binding activity.     

The functional units of a peptostreptococcal protein L

Protein L is a cell-surface protein from Peptostreptococcus which interacts with immunoglobulin kappa light chains. A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof were expressed in Escherichia coli. The peptides were examined for binding to immunoglobulin and serum albumin. The four C units were shown to be responsible for binding to immunoglobulin and the four D units for binding to albumin. This protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. The albumin-binding units have high amino acid sequence identity with the albumin-binding units of streptococcal cell-surface proteins. The gene contains three sites available for internal initiation of translation resulting in three active proteins. The protein L molecule presented in this report was compared with a previously reported protein from Peptostreptococcus strain 312. The two proteins differ in several respects, including size and the number and types of repeat units.     

Platelet activating factor synthesis and metabolism in intestinal ischaemia-reperfusion injury

The object of this study was to characterize the synthesis and metabolism of platelet activating factor (PAF) by intestinal mucosa subjected to ischaemia-reperfusion injury. Canine intestinal mucosa produced 16:0-PAF, 18:0-PAF, and high levels of the corresponding lyso- PAF metabolites. Three h of intestinal ischaemia and ischaemia followed by 1 h of reperfusion did not affect the synthesis or metabolism of PAF by intestinal mucosa. Intestinal mucosa elaborated a factor that rapidly hydrolyzes PAF to lyso-PAF. The observed hydrolysis rate was not altered by ischaemia or ischaemia and reperfusion. In conclusion, this study suggests that intestinal mucosa produces PAF and rapidly hydrolyzes PAF. The PAF synthesis and metabolism rates of intestinal mucosa is not altered by ischaemia reperfusion in this model under the imposed conditions.     

Human T-lymphotropic virus type I seroprevalence among Japanese Americans.

The epidemiology of human T-lymphotropic virus type I (HTLV-I) infection is not well defined in Japanese Americans. This impairs using approaches that could reduce viral transmission and monitor carriers for the disease. Using enzyme-linked immunosorbent assay and p21e recombinant Western blot testing, HTLV-I antibody was measured in unlinked samples from Japanese-American patients at 4 physicians'' offices in San Francisco, California. Of 442 patients, 4 (0.9%; 95% confidence interval 0.25%, 2.3%) were confirmed seropositive, all with an HTLV-I rather than an HTLV-II pattern on Western blot. Seroprevalence was highest among the issei or immigrant generation (3/230 or 1.3%) compared with the second-generation nisei (1/191 or 0.5%) or third-generation sansei (0 of 17). Prevalence did not differ by age or sex, although the number of positive subjects in each subgroup was small. Of 88 patients with familial origins in endemic areas of southern Japan, none were seropositive. In this sample of Japanese Americans, HTLV-I seroprevalence was lower than in residents of endemic southern Japan but higher than among American blood donors. The prevalence was most similar to that in nonendemic areas of Japan. The public health implications of HTLV-I infection among Japanese Americans are discussed.     

Mechanisms of TonB-catalyzed iron transport through the enteric bacterial cell envelope

The recent solution of enteric bacterial porin structure, and new insights into the mechanism by which outer membrane receptor proteins recognize and internalize specific ligands, advocates the re-evaluation of TonB-dependent transport physiology. In this minireview we discuss the potential structural features of siderophore receptors and TonB, and use this analysis to evaluate both existing and new models of energy and signal transduction from the inner membrane to the outer membrane of gram-negative bacteria.     

Properties of Escherichia coli expressing bacteriophage P22 Abc (anti-RecBCD) proteins, including inhibition of Chi activity.

Escherichia coli strains bearing plasmids expressing phage P22 anti-RecBCD functions abc1 and abc2 were tested for the presence of recBC-like phenotypes. Abc2 induces moderate sensitivity to UV light in wild-type and recD mutant strains but severely sensitizes both recF and recJ mutants. Abc1 has little effect on UV sensitivity in wild-type or recF or recJ mutant hosts but increases the sensitivity of recD mutants to a UV dose of 20 J/m2 about 10-fold. Abc2 induces E. coli to segregate inviable cells during growth, interferes with the growth of lambda red gam chi+ and chi 0 phage (the effect is greater with chi+ phage), inhibits Chi and Chi-like activity as measured by lambda red gam crosses, and prevents SOS induction in response to nalidixic acid; Abc1 has no effect in these tests. Abc2, alone or with Abc1, does not allow the growth of lambda red gam in the presence of a P2 prophage but does not kill the P2 lysogenic host (as lambda Gam does). Finally, Abc2 inhibits conjugational recombination in wild-type cells to the level seen in recBC mutants. These data suggest that Abc2 inhibits the recombination-promoting ability of RecBCD but leaves the exonuclease functions intact.