Novel non-isotopic method for the localization of receptors in tissue sections.

We describe a novel fluorescent method for the detection of receptors for chimeric proteins in tissue sections. The technique was developed using a recombinant human insulin-like growth factor (IGF-1) chimera, bearing six additional histidine residues at the carboxy-terminal end (IGF-1-His). We demonstrated that dehydration of the tissue sections was detrimental for binding and that its prevention dramatically increased sensitivity. The specificity of IGF-1-His interaction was shown by gradual abolition of the fluorescent signal in the presence of increasing concentrations of IGF-1. Combining immunofluorescence with in situ ligand binding, we showed that IGF-1-His binding corresponded to the IGF-1 receptor (IGFR-1) distribution in human fetal kidney. Moreover, incubation of the tissue sections with an anti-IGFR-1 blocking antibody abolished IGF-1-His binding, demonstrating that the interaction was mediated by the IGFR-1. The method was also used to localize the IGFR-1 in E18 rat embryo sagittal sections. The IGF-1-His binding pattern was observed in brain, cartilage, lung, skin, heart, diaphragm, and tongue, and paralleled the previously reported IGFR-1 distribution. We believe that this new non-isotopic in situ ligand binding method will facilitate rapid and accurate localization of receptors in tissue sections.     

Expansion of repetitive DNA into cytogenetically visible elements.

Two cases of amplified repetitive elements accidentally identified in cancer samples are reported. In both cases, repeated DNA that is normally not visible by traditional chromosome banding had increased in amount to become cytogenetically visible. In one case, an addition to the short arm of chromosome 1 was originally diagnosed. However, upon molecular analysis the diagnosis could be corrected to an amplification of the D1Z2 repeat. In the second case, a strongly DAPI-positive band was visible at the top of the short arm of chromosome 22, and the original diagnosis was add(22). Staining for telomeric repeats revealed their presence inside the DAPI-positive element, thus confirming that the element in question was truly added to the end of the chromosome. Curiously, no telomeric repeats could be detected distal to the DAPI-positive element. The identity of the DAPI-positive element could not be established, as it was not stained by any of the specific probes applied, nor in a scanning hybridization with labeled Cot-1 DNA. It thus seems to represent an expansion from some lowly repetitive AT-rich DNA translocated to the tip of chromosome 22.     

Purification of tryptic peptides for mass spectrometry using polyvinylidene fluoride membrane.

A simple procedure for the purification of tryptic peptides, prior to mass spectrometric analysis, using polyvinylidene fluoride membrane (PVDF) is described. The sensitivity of mass spectrometric analysis is such that minor impurities in tryptic peptide digests suppress the signal obtained. However, we obtained useful signal, from a sample that did not yield any spectra earlier, by purifying the sample using PVDF membrane. For this, the tryptic peptide digest was first spotted on the membrane which was then air-dried and washed. Further, the membrane was extracted with trifluoroacetic acid (TFA) and acetonitrile and subjected to mass spectrometric analysis. This procedure enabled us to identify a cross-reactive D1 antigen on the neutrophil surface that bound antibodies that targeted 60 kD Ro autoantigen in systemic lupus erythematosus, an autoimmune disorder.     

Influence of food motivation on neuronal response of the cat somatic cortex during instrumental reflex

Spike activity in neurons of areas 3 and 4 was investigated in experiments on cats during the conditioned reflex response of placing the paw on a support both before and after feeding ad libitum. Ingestion of a feed consisting of a rapidly absorbed glucose dairy mix did not prevent the reflex taking place if the animals' favorite food was used as reinforcement. Background activity increased in two-thirds of the neurons after the feed; the tonic constituent of neuronal response declined substantially and repeated contraction of the biceps occurring at the same rate as locomotor movements disappeared. Difference in latency of response produced by the conditioned stimulus in the same neurons before and after feeding measured 50–300 msec during the experiment. Measurements of latency of placing motion remained largely unchanged. Changes in the latency of neuronal spike response were thus found to be interrelated with the intensity of the animal's motivational excitation. It is suggested that fluctuations in degree of food motivation lead to changes in cortico-subcortical relationships responsible for initiation and performance of conditioned movements in these animals.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 5, pp. 646–653, September–October, 1987.     

L-glutamate activated membrane conductivity in spinal cord neurons during early chick embryogenesis

Transient and steady-state components of L-glutamate-activated membrane currents were investigated using intracellular perfusion, voltage clamp, and concentration clamp techniques in spinal cord neurons of 6–11 day chick embryos. Hill's coefficient was found to equal 1 for transient and 2 for steady-state components. It was shown that the L-glutamate-activated receptors are present, which appear in the membrane of spinal neurons at the early stages of development.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 2, pp. 251–258, March–April, 1987.     

Isolation of Actinobacillus suis from a Canada goose (Branta canadensis)

Blindness from conjunctivitis caused by Actinobacillus suis was found in a Canada goose (Branta canadensis) from a wildlife refuge in Kentucky. Lesions were not observed elsewhere and other body organs were grossly normal. Actinobacillus spp. in birds is rare and this is apparently the first report of conjunctivitis resulting from this infection in waterfowl.     

Vaginal treatment with a prostaglandin F2 alpha derivative (alfaprostol) for inducing labor in pregnancy at term

The effectiveness and acceptability of Alfaprostol (an analog of PGF2 alpha) in inducing labor were assessed in 20 pregnant women at term. All subjects had no spontaneous uterine activity before treatment and the mean (M +/- SE) Bishop score was 2.45 +/- 0.21. The drug was administered by vaginal route at the dose of 10 mg every 3 hours. Regular uterine contractions appeared in all patients and delivery occurred in 85% of the patients after a mean time of 9h50min +/- 0h55min following the start of treatment. The mean dose of Alfaprostol utilized to achieve delivery was 29.4 +/- 2.0 mg. No major side effects were noted in the mothers and their fetuses at any time during treatment. Two patients exhibited vomiting. The Apgar score of all newborns at birth was 8 or more. These results suggest the usefulness of Alfaprostol to induce labor in pregnant women at term, as it has oxytocic activity without adverse effects on either the mother or the fetus.     

Critical considerations in the development of an assay for sulfidopeptide leukotrienes in plasma

A sensitive and specific assay has been developed for measurement of total sulfidopeptide leukotrienes (LT) in plasma. LTC4 and LTD4 in plasma are converted to LTE4 which is then extracted by C18 Sep-Pak binding and elution. Total LTE4 is resolved by reverse phase high performance liquid chromatography (RP-HPLC) and quantitated by radioimmunoassay (RIA). A [3H]LTE4 internal standard is added to the starting plasma sample to allow overall recovery to be calculated and to define the fractions from RP-HPLC to be assayed for LTE4-like immunoreactivity. The correlation between the measured increase in LTE4 concentration after addition of incremental amounts of LTC4 and LTE4 to plasma was 0.989 and 0.978, respectively, with slopes of 1.05 and 1.11. Addition of 51 pg/ml LTE4 to 5 ml plasma was detectable; the measured increase was 48 +/- 12 pg/ml (mean +/- SE, n = 7). The intra-assay coefficient of variation for 341 pg/ml of added LTC4 was 3.2% (n = 6). Sulfidopeptide leukotrienes could not be detected in blood samples taken from 12 normal volunteers in whom the theoretical detection limit, calculated from the sensitivity of the RIA, the overall recovery of LTE4, and the volume of plasma extracted, was 83 +/- 4 pg LTE4/ml plasma (0.19 +/- 0.01 pmol sulfidopeptide leukotriene/ml plasma; mean +/- SE).     

The effect of Ca2+ on the thermal stability of trypsin in phosphate-buffered saline solution used for harvesting of human embryonic lung fibroblast cultures

The stabilizing effect of Ca2+ (264.9 mg CaCl2 X 2H2O ml-1) on a 0.5% solution of twice-crystallized bovine trypsin in phosphate-buffered saline (used for harvesting human embryonic lung fibroblasts) was studied at 7-37 degrees C and at -20 and -70 degrees C. It can be concluded that storage of the enzyme in the buffer (with or without Ca2+) is not advisable at temperatures greater than or equal to 20 degrees C. At 7 degrees C, on the other hand, trypsin can be stored for some weeks in the calcium-containing phosphate-buffered saline if a moderate loss of activity is acceptable. At -20 degrees C and -70 degrees C the stability of the enzyme was good. In the presence of Ca2+ about 90% of the activity remained after 18 weeks. Without Ca2+ the activity was approximately 10% lower.     

An abnormal terminal X-Y interchange accounts for most but not all cases of human XX maleness

To determine if human XX maleness results from an abnormal chromosomal X-Y interchange, we studied the inheritance of the paternal pseudoautosomal region in nine patients. Those six patients in whom Y-specific DNA was found (Y(+)) inherited the entire pseudoautosomal region from the paternal Y chromosome and lost that of the paternal X chromosome. Moreover, in three Y(+) cases, we observed the deletion of a paternal Xp locus tightly linked to the pseudoautosomal region. These results definitively show that an abnormal and terminal X-Y interchange during paternal meiosis causes Y(+)XX maleness. In contrast, no abnormal X-Y interchange was observed in any of the three Y(-) cases analyzed, suggesting that maleness can occur in the absence of any Y-specific DNA.