Comparison of the properties of a soluble form of glucocerebrosidase from human urine with those of the membrane-associated tissue enzyme

Human urine contains a soluble form of glucocerebrosidase, an enzyme associated with the lysosomal membrane in cells and tissues. Urinary glucocerebrosidase is identical to the enzyme extracted from tissues with respect to the following parameters: Km for natural and artificial substrates, inhibition by conduritol B-epoxide, and stimulation by taurocholate. The enzyme is greater than 90% precipitable by polyclonal anti-(placental glucocerebrosidase) antiserum. Upon isoelectric focussing of urinary glucocerebrosidase multiple peaks of activity were observed. Partial deglycosylation (removal of sialic acid, N-acetylglucosamine and galactose) of the urinary enzyme increased the isoelectric point to a value identical to that of the main form found after partial deglycosylation of the placental enzyme. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate followed by immunoblotting, the immunopurified urinary enzyme shows the same molecular mass forms as the enzyme immunopurified from brain and kidney. In placenta the apparent molecular mass is somewhat higher but upon removal of sialic acid, N-acetylglucosamine and galactose the urinary and the placental enzyme show identical molecular masses of 57 kDa. We conclude that the enzymes extracted from urine and tissue are identical and that differences in apparent molecular mass and isoelectric point are probably due to heterogeneity in the oligosaccharide moieties of the molecules.     

Effect of long-term Intralipid administration in mice

Intralipid was administered intravenously to mice at a level of 2 g kg-1 day-1 for 23 days. No alterations in phagocytic index, liver or spleen size were observed in the chronically injected mice as compared with control mice that received saline injections. Tissue distribution of 0.45 micron multilamellar liposomes of egg phosphatidylcholine:cholesterol (2:1) was similar in mice that had been chronically injected with Intralipid to that in control mice. Mice chronically given the same total amount of phospholipid in the form of 0.2 micron liposomes of phosphatidylcholine:cholesterol (2:1) rather than as a lipid-triglyceride emulsion showed altered tissue distribution of entrapped label with decreased liver uptake and increased splenic uptake, which is indicative of reticuloendothelial blockade. Tissue distribution of [14C]dipalmitoylphosphatidylcholine Intralipid was compared with that of [14C]dipalmitoylphosphatidylcholine 0.2 micron MLV of phosphatidylcholine:cholesterol (2:1). Intralipid was taken up 2- to 3-fold less by liver and 5- to 10-fold less by spleen than liposomes. Blood levels of Intralipid were higher than those of liposomes. [14C]dipalmitoylphosphatidylcholine Intralipid was eliminated from the body at a faster rate than [14C]dipalmitoylphosphatidylcholine liposomes. The lack of reticuloendothelial blockade caused by Intralipid as compared with liposomes appears to be related to its diminished uptake into reticuloendothelial tissues. This diminished uptake may be related to differences in apolipoprotein uptake of Intralipid, which is primarily in the form of a phospholipid monolayer, and liposomes, which have their phospholipid organized into a bilayer.     

Symbiotic Relationship of Bacteroides cellulosolvens and Clostridium saccharolyticum in Cellulose Fermentation

In coculture, Bacteroides cellulosolvens and Clostridium saccharolyticum fermented 33% more cellulose than did B. cellulosolvens alone. Also, cellulose digestion continued at a maximum rate 48 h longer in coculture. B. cellulosolvens hydrolyzes cellulose and supplies C. saccharolyticum with sugars and a growth factor replaceable by yeast extract. Alone, B. cellulosolvens exhibited an early cessation of growth which was not due to nutrient depletion, low pH, or toxic accumulation of acetic acid, ethanol, lactic acid, H₂, CO₂, cellobiose, glucose, or xylose. However, a 1-h incubation of B. cellulosolvens spent-culture medium with C. saacharolyticum cells starved for growth factor allowed a resumption of B. cellulosolvens growth. The symbiotic relationship of this naturally occurring coculture is one of mutualism, in which the cellulolytic microbe supplies the saccharolytic microbe with nutrients, and in turn the saccharolytic microbe removes a secondary metabolite toxic to the primary microbe.     

Polyploid cells in blastocysts and early fetuses from Australian Merino sheep

Cytogenetic examination was made of 103 13-14-day-old blastocysts and 116 24-32-day-old fetuses from untreated and androstenedione-7-HSA-immunized Merino ewes. There were no differences in the chromosome composition of blastocysts or fetuses from treated or untreated ewes and so the data were combined. At Days 13-14 a 1N/2N mosaic and a 2N - 1/2N/4N mosaic embryo were observed. In addition, 52 of the blastocysts were 2N/4N mosaics, with 8 of these also containing 8N cells, and one blastocyst was a 2N/8N mosaic. No aneuploid fetuses were observed, but 80 of the 116 fetuses contained polyploid cells, including 4N, 6N and 8N cells. The polyploid cells observed in the blastocysts and fetuses should not be considered as abnormal cells as they appear to be a normal part of the developmental processes leading to trophoblast formation and fetal differentiation.     

The detection and purification of a cat uterine secretory protein that is estrogen dependent (CUPED)

An estrogen-dependent secretory protein (CUPED) was detected and purified from uterine flushings of ovariectomized cats treated with 17 beta-estradiol. The protein was not detected in uterine flushings obtained from untreated ovariectomized animals or estrogen-primed animals treated with progesterone for 4 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of uterine flushings showed the presence of 1 or 2 protein bands with relative mobility values less than reduced and denatured thyroglobulin (Mr = 330,000). The protein was purified by differential centrifugation and gel filtration chromatography. Antiserum was raised against this purified protein in rabbits. The specificity of the antiserum to uterine fluid proteins was assessed by immunoblotting of electrophoretically transferred proteins. The antiserum cross-reacted with electrophoretically separated CUPED protein bands in uterine flushings. This protein may represent the content of the estradiol-induced secretory granules present in endometrial epithelial cells.     

Modulation of collagen production in cultured fibroblasts by a low-frequency, pulsed magnetic field

Primary cultures of chicken tendon fibroblasts have been exposed for various periods to a low-frequency, pulsed magnetic field, and the effects on protein and collagen synthesis have been examined by radioisotopic incorporation. Total protein synthesis was increased in confluent cells treated with a pulsed magnetic field for the last 24 h of culture as well as in cells treated for a total of 6 days. However, in 6 day-treated cultures, collagen accumulation was specifically enhanced as compared to total protein, whereas after short-term exposure, collagen production was increased only to the same extent as total protein. Levels of cyclic AMP were significantly decreased after 6-day pulsed magnetic field treatment, probably as a consequence of diminished adenylate cyclase activity. Exposure to pulsed magnetic field had no effect on cell proliferation or collagen phenotype. These results indicate that a pulsed magnetic field can specifically increase production of collagen, the major differentiated function of fibroblasts, possibly by altering cyclic-AMP metabolism.     

A charge coupled device-based image cytophotometry system for quantitative histochemistry and cytochemistry

A rapid, semiautomated cytophotometry system for quantitative histochemistry and cytochemistry was constructed. The system consists of a Fairchild charge coupled device (CCD) image camera, a Zeiss Universal microscope, a Datacube analog to digital converter, and a digital Equipment Corporation LSI 11/23 computer operating under RT-11. Computer programs were written in FORTRAN and the MACRO assembly language for the acquisition of data from the CCD device. These data were then used for image segmentation, image display, and calculation of total optical density, perimeter, cell area, and several shape features. The reproducibility of measurement made with the CCD-based cytophotometry system was tested by repeated measurements. The coefficient of variation was estimated to be 1.7% for total optical density and 0.9% for cell area. The CCD-based cytophotometry system was further evaluated by comparing results with measurements made on the same cells with a scanning stage cytophotometer using the HIDACSYS computer programs. Correlation coefficients of 0.96 for total optical density and 0.91 for cell area were obtained between the two systems. We conclude that the high-speed, dimensional stability, small size, and linearity of the CCD-based cytophotometry system will make it useful for quantitative histochemistry and cytochemistry.     

Thymosin fraction 5 causes increased serum corticosterone in rodents in vivo

In these studies it was found that i.p. injection of thymosin fraction 5 (TF5) caused a dose-dependent increase in serum corticosterone in male Swiss Webster mice and in male Wistar rats. The maximum responses were seen at 1 and 2 hr, respectively. There was no effect on serum corticosterone in mice when Thymosin alpha 1 (a 28 amino acid peptide isolated from TF5) was injected i.p. at doses up to 100 micrograms. The steroidogenic effects of TF5 were seen only when the basal levels of serum corticosterone were low (less than 80 ng/ml). In studies in which the baseline levels in the animal colony were elevated (greater than 80 ng/ml), there were no steroidogenic effects, or they were minimal. These results suggest that some component of TF5 may influence pituitary adrenal function.     

Characterization of a T4+/Leu-8+ T cell clone that directly helps B cell Ig production by secreting B cell differentiation factor

A human T4+/Leu-8+ T cell clone (YA2) was established by phytohemagglutinin activation and interleukin 2 (IL 2) propagation. Functional characterization of this clone demonstrated that it provided potent help towards Ig production by pokeweed mitogen-stimulated B cells in the presence of small numbers of autologous T cells or by Staphylococcus aureus Cowan I (SAC)-activated B cells in the presence of B cell growth factor (BCGF). YA2 provided no help to resting B cells and minimal help to either unactivated B cells cultured with BCGF or SAC-activated B cells. Supernatant generated from clone YA2 by IL 2 stimulation had significant B cell differentiation activity but no BCGF or IL 2 activity. Thus, YA2 is a T4+/Leu-8+ potent direct helper only to B cells that are activated and proliferating due to its selective secretion of a differentiation factor, and not an activation and growth factor. The availability of phenotypically defined cloned populations of T cells with restricted functional helper activity related to the secretion of selected B cell tropic factors should prove useful in the dissection of the role of individual T cell subsets in the regulation of the human B cell cycle.     

Long-term infertility and dominant lethal mutations in male mice treated with adriamycin

Sperm production and fertility were studied in male mice treated with adriamycin (ADR) at 6 or 8 mg/kg. Testicular sperm production and epididymal sperm counts were markedly reduced after ADR treatment. Gradual recovery of counts occurred, but sperm counts had not reached control levels even more than 1 year after treatment. Epididymal sperm showed treatment-induced morphological abnormalities throughout the experiment; the frequencies of sperm with detached tails and the frequencies of sperm with morphologically abnormal heads remained elevated about 2-3-fold above control. According to the frequency of vaginal plugs, treated male mice mated at control rates with untreated females during the post-treatment sterile period. However, after some fertility was regained the fertilization rate (calculated as the fraction of eggs, flushed from the oviduct 2 days after mating, that had been fertilized and had cleaved) was markedly reduced and remained depressed for the remainder of the experiment. The fertilization rate reached only 0.29 at 23-32 weeks after 8 mg/kg ADR and 0.76 at 16-23 weeks after 6 mg/kg ADR; both values were significantly below the control value of 0.94. Dominant lethal mutations in the zygotes flushed from the oviduct were measured in culture by the loss of the zygote's ability to develop to a stage characterized by trophectoderm outgrowths and formation of an inner cell mass. The frequencies of dominant lethal mutations detected in vitro were 1.7 or 7.4% after 6 mg/kg, and 32 or 40% after 8 mg/kg ADR; each value was calculated in two different ways, with 3 of these 4 values significantly different from zero. We conclude that even after mice regain fertility following ADR exposure, the level of fertility remains permanently subnormal as evidenced by a lack of fertilization of eggs that is probably due to the decreased quantity and quality of spermatozoa produced. Furthermore, ADR can induce genetic damage in stem spermatogonia, which can be transmitted through fertile spermatozoa. Thus, there may be a genetic risk to the offspring of cancer patients treated with ADR chemotherapy, but at present we are unable to quantitate that risk.