Basal gonadotropin hormone release rates during the period of selective follicle-stimulating hormone release in the juvenile female rat

There are situations in which adult female rats release increased amounts of follicle-stimulating hormone (FSH) independent of increased luteinizing hormone (LH) release. This results from, at least in part, a selective increase in the basal FSH release rate. We investigated whether an increase in the basal FSH release rate is contributory to the rise in serum FSH levels which occurs independent of a rise in serum LH levels in the immature female rat. Rats had high serum FSH concentrations on days 7 and 15 after birth, low serum FSH levels on day 23, and low serum LH levels on all three days. In contrast, anterior pituitary gland (APG) FSH and LH concentrations and contents increased from day 7 to day 15 and the contents increased further from day 15 to day 23. Similarly, basal FSH and LH release rates per mg APG or per APG, as assessed by measurement of FSH and LH released into culture medium containing APG(s) from different aged rats, increased from day 7 to day 15 but did not increase further between days 15 and 23. The results indicate that unlike situations observed to date in adult female rats, a mechanism(s) other than an increase in the basal FSH release rate is involved in selective FSH release in the immature female rat.     

Clinical usefulness of estimation of serum fructosamine concentration as a screening test for diabetes mellitus.

Fructosamine, a putative measure of serum glycosylated proteins, was measured in 74 subjects referred for oral glucose tolerance tests. A normal range (mean (2 SD] of 1.6 (0.4) mmol/l (40(10) mg/100 ml) derive from results obtained in 83 healthy non-diabetic volunteers permitted the detection of 15 out of 17 (88%) subjects with proved diabetes and yielded only five (9%) false positive diagnoses. Fructosamine concentrations correlated significantly (p less than 0.001) with fasting plasma glucose concentrations (r = 0.76) and glycosylated haemoglobin concentrations (r = 0.70). A longitudinal study suggested that fructosamine concentration was an index of intermediate term (one to three weeks) blood glucose control. Fructosamine concentration was not related to uraemia and did not depend on albumin or total protein concentrations, provided that serum albumin concentrations remained above 30 g/l. Estimation of fructosamine concentrations is a fully automated procedure and may provide a simple means of screening for diabetes mellitus.     

Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells

When granulocyte colony-stimulating factor (G-CSF), purified to homogeneity from mouse lung-conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G-CSF, a small proportion of macrophage and granulocyte-macrophage colonies also developed. G-CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence-activated cell sorter, indicating that G-CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G-CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G-CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte-macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G-CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G-CSF was mixed with purified granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), the combination stimulated the formation by adult marrow cells of more granulocyte-macrophage colonies than either stimulus alone and an overall size increase in all colonies. G-CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM-CSF.     

Receptors for C3 and IgG on macrophage, neutrophil and eosinophil colony cells grown in vitro.

Macrophage, neutrophil, and eosinophil colony cells from bone marrow culture in semisolid agar medium were studied for membrane C3 and IgG receptors. The capacity of these cells to bind either erythrocytes-19S antibody-complement (EAC) or erythrocyte-7S antibody (EA7S) complexes was measured using the rosette method. Whereas macrophage and neutrophil colony cells showed receptors for both C3 and IgG, eosinophil colony cells appear to bear only IgG receptors. Studies correlating colony age and the presence of receptors showed that 60 to 70% of the cells from 3-day-old macrophage colonies were reactive for EAC and EA7S contrasting with 80 to 90% of the cells from 6- to 12-day-old colonies. Neutrophils behaved somewhat differently: EAC and EA7S reactive cells were seen in colonies after 4 or 5 days in culture and comprised only 50 to 60% of the colony population. Eosinophilic colonies showed 50 to 60% EA7S reactive cells after 6 to 7 days in culture, but no EAC reactive cells were found among these colonies at any time. The characteristics and properties of the receptors detected on colony cells were similar to those on macrophages and neutrophils from normal peritoneal fluid or bone marrow. Most macrophage colony cells were actively phagocytic whereas neutrophils and eosinophilic colony cells failed to show phagocytosis under the same conditions.     

Virus in Water. II. Evaluation of Membrane Cartridge Filters for Recovering Low Multiplicities of Poliovirus from Water

The efficiency of a Millitube MF cartridge filter, a membrane filter, for recovery of poliovirus from 100-gal volumes of both fresh (tap) and estuarine water was determined. In the high multiplicity of virus input-output experiments, recovery of 97% or greater of input virus was achieved in both types of water when the final concentration of divalent cation as Mg(2+) was 1,200 mug/ml and the pH was 4.5. Virus was effectively eluted from the membrane cartridge with 5x nutrient broth in 0.05 M carbonate-bicarbonate buffer at pH 9.0. Four elutions of 250 ml each were used. In the low multiplicity of virus input-output experiments under the same cationic and pH conditions, up to 67% of the input virus was recovered when the virus was further concentrated from the eluates by the aqueous polymer two-phase separation technique. The volume reduction was 126,000-190,000 to 1. The use of the combined techniques, i.e., membrane adsorption followed by aqueous polymer two-phase separation, provided a highly sensitive, simple, and remarkably reliable sequential methodology for the quantitative recovery of poliovirus occurring at multiplicities as low as 1 to 2 plaque-forming units per 5 gal of water.     

Staphylococcus aureus Response to Lysostaphin in Some Fermented Foods

The addition of lysostaphin to starting materials for cheese and fermented sausage that were artificially contaminated with Staphylococcus aureus resulted in an initial decrease in the staphylococcal flora. In a simulated cheese process, lysostaphin remained with the curd after separation of the whey. In both cheese and fermented sausage samples that were produced experimentally in the laboratory, a significant S. aureus population ultimately developed, even in the presence of lysostaphin. Staphylococcal isolates from these treated products were not more resistant to the lytic enzyme than was the parent strain.     

In vitro actions on hemopoietic cells of recombinant murine GM-CSF purified after production in Escherichia coli: comparison with purified native GM-CSF

Recombinant murine GM-CSF produced in Escherichia coli was purified to homogeneity and tested in parallel with purified native GM-CSF. Both recombinant and native GM-CSF stimulated granulocyte and/or macrophage colony formation by adult and fetal mouse progenitor cells, and with adult marrow cells the specific activity of the recombinant GM-CSF (25 X 10(8) U/mg) was similar to that of the native form (15 X 10(8) U/mg). At high concentrations (greater than 200 U/ml), both forms of GM-CSF also stimulated eosinophil colony formation by adult marrow cells and, at very high concentrations (greater than 800 U/ml), megakaryocyte and some erythroid and mixed-erythroid colony formation. Recombinant GM-CSF was as effective in stimulating the proliferation of the GM-CSF-dependent cell line FD as the native molecule. Both recombinant and native GM-CSF were able to induce partial differentiation in colonies of WEHI-3B myeloid leukemic cells. Recombinant GM-CSF competed effectively for the binding of 125I-labeled native GM-CSF to hemopoietic cells, and antiserum to recombinant GM-CSF also neutralized the biological activity of native GM-CSF. The bacterially synthesized GM-CSF was a slightly more effective stimulus for megakaryocyte colony formation than the native molecule. The demonstration that purified bacterially synthesized GM-CSF is biologically active in vitro now permits studies to be undertaken on the in vivo effects of this material.     

Granulocyte/macrophage colony-stimulating factor from mouse lung conditioned medium. Purification of multiple forms and radioiodination.

Four forms of mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) were purified 100,000-fold from mouse lung conditioned medium. Each of the CSF species stimulated the formation of both granulocyte and macrophage colonies, and half-maximal stimulation in the semi-solid mouse bone-marrow colony assay occurred at 1 pm. The four GM-CSF species exhibited similar charge microheterogeneity, focusing between pH 4.2 and pH 5.2. On SDS/polyacrylamide gels two of the GM-CSF sub-species had apparent Mr values of 23,000, and the other two, 21, 000. Treatment with neuraminidase decreased the Mr values of these two sets to 21,000 and 19,000 respectively. Incubation with endoglucosidase F decreased the charge heterogeneity and the Mr of all species to 16,500. A gas-phase radioiodination procedure was used to incorporate 2-3 atoms of 125I/molecule into purified GM-CSF without any loss of biological activity. The 125I-labelled GM-CSF was analysed on a microbore reversed-phase h.p.l.c. column to determine its specific radioactivity directly. This 125I-labelled GM-CSF molecule is suitable for cell-surface receptor-binding studies.     

Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes.

The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10(4) physical particles of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hybridization stringency, 32P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments.     

Improved method and test strategy for recovery of enteric viruses from shellfish

An improved recovery method and testing strategy were devised for recovery of low numbers of enteric viruses from each of three commercially important shellfish species. Effective recovery of virus depended as much upon details of the test strategy adopted for use of the improved method with each species as on the method itself. The most important test details involved sample composition, pool size, and method of use of cell cultures. Recovery sensitivity measured permitted detection of 25 to 3 plaque-forming units of enteroviruses and 100 to 27 plaque-forming units of reovirus through their recovery in cell culture, with effectivenesses averaging 64 and 46%, respectively. Test samples prepared by the improved recovery method were virtually cytotoxicity free. Optimal recovery of virus on 45-cm2 cell culture monolayers was obtained with 1-ml inocula adsorbed for 2 h. The most effective recovery of virus from shellfish samples was made by a sequential adsorption procedure which allowed equal exposure of an entire sample to each of two or more cell cultures. Removal of nonviral contaminants from test samples by antibiotic treatment was preferable to the use of ether or membrane filtration procedures.