Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter

One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80^o C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.     

Unit activity in the posterior sigmoid and proreal gyri of the cat cortex during bemegride administration

Unit activity was studied in the posterior sigmoid and proreal gyri of the cat cortex in response to photic, acoustic, and somatic stimulation. The animals were first anesthetized with pentobarbital and, after recovery, were given intravenous injections of bemegride (β-ethyl-β-methylglutarimide) and immobilized with diplacin [1, 3-di(β-platyneciniumethoxy)-benzene hydrochloride]. The state of the preparation used was similar to the state of waking animals as regards the characteristics of its unit activity. Unit responses of the proreal gyrus were chiefly modally nonspecific and consisted of brief suppression of spontaneous activity for 100–200 msec. Neurons of the posterior sigmoid gyrus were characterized by much greater modal specificity and variety of types of responses to stimuli of different modalities. Comparison of the characteristics of unit activity in these parts of the neocortex suggests that the frontal cortex in cats is still a highly unspecialized structure.     

The cystic fibrosis transmembrane conductance regulator (CFTR): three-dimensional structure and localization of a channel gate

Cystic fibrosis affects about 1 in 2500 live births and involves loss of transmembrane chloride flux due to a lack of a membrane protein channel termed the cystic fibrosis transmembrane conductance regulator (CFTR). We have studied CFTR structure by electron crystallography. The data were compared with existing structures of other ATP-binding cassette transporters. The protein was crystallized in the outward facing state and resembled the well characterized Sav1866 transporter. We identified regions in the CFTR map, not accounted for by Sav1866, which were potential locations for the regulatory region as well as the channel gate. In this analysis, we were aided by the fact that the unit cell was composed of two molecules not related by crystallographic symmetry. We also identified regions in the fitted Sav1866 model that were missing from the map, hence regions that were either disordered in CFTR or differently organized compared with Sav1866. Apart from the N and C termini, this indicated that in CFTR, the cytoplasmic end of transmembrane helix 5/11 and its associated loop could be partly disordered (or alternatively located).     

Hydrolysis of chimeric proteins by enteropeptidase at the specific linker (Asp)4Lys depending on refolding conditions

Refolding from inclusion bodies of chimeric proteins containing the enteropeptidase-specific linker (Asp)4Lys was carried out. It was shown that, depending on the refolding conditions, chimeric proteins function as substrates or inhibitors of the enteropeptidase. The efficiency of the enteropeptidase hydrolysis of chimeric proteins containing the (Asp)4Lys linker may depend not only on the amino acid sequence of the protein binding site for the enzyme but also on the site conformation.     

Ranging patterns of two syntopic gerbillid rodents: a radiotelemetry and live-trapping study in semi-desert habitat of Kalmykia,Russia

We studied ranging patterns of syntopic folivorous and granivorous gerbils (Meriones tamariscinus Pallas, 1773 andMeriones meridianus Pallas, 1773, respectively) by means of radiotracking and live-trapping in Kalmykia, southern Russia. We hypothesized that ranging patterns exhibited by these rodents would differ in accordance with differences in their preferred diets. Trapping and radiotracking were performed from 23 May to 20 June 2000 on a 12 ha grid divided into 20 × 20 m squares. To assess ranging patterns we estimated the number of grid squares visited by an individual, 100% minimum convex polygon, mean squared distance from the center, maximal distance between individual locations, linearity index, and distance measures at various temporal scales. In accord with expectations, females of folivorousM. tamariscinus exhibited smaller ranges than females of granivorousM. meridianus. Contrary to our expectations both trapping and radiotracking data revealed similar “granivorous” ranging patterns in males of both species of gerbils. MaleM. meridianus andM. tamariscinus were similar in number of burrows used, home range size, and distances traversed. No temporal dependence of distance measures was revealed in either species; males of both species traversed similar distances regardless of time interval for distance measures so that single-night movements and multiday movements did not differ in either species. High food selectivity in maleM. tamariscinus is considered to be responsible for the “granivorous” pattern of space use in this folivorous species.     

Visual afferentation deficit during diffuse photosensitivity period in nestlings affects morphogenesis in Wulst neurons

Golgi-stained preparations were processed with computerized morphometry to study the effect of the deprivation (eyes covered with nontransparent caps) on the development of neurons in the Wulst (the structure analogous to mammalian visual cortex) of pied flycatcher nestlings. Six-day-old nestlings that have only one form of the visual perception--diffuse photosensitivity--were studied. In the previous paper [Korneeva et al., 1994] in was shown that the Wulst of 6-day-old nestlings consisted of non-differentiated neurons and stellate-like cells at different stages of maturation; the latter group was subdivided into more- and less-mature cells. This work proved that even a 1.5-day-long deprivation (actual duration being counted off from the moment of appearance of the first retinal photoreceptors at the age of 4.5 days) resulted in significant changes in the geometry of stellate-like neurons. The changes in less-mature cells were predominantly destructive (decline of all quantitative indices of a cell, including the significant decrease in the total length of dendrites, maximal radius of the dendritic field and cell branching index), while the changes in more-mature cells were constructive (increase in all quantitative cell indices, including statistically significant increase in the soma section area, total length of dendrites, maximal radius of dendritic field and the number of foci of maximal branching). Different reactions of these cell types to the limitation of visual afferentation may be connected with differences in the afferent inflow to less- and more-mature cells or/and with different maturational stages of these neurons at the onset of deprivation.     

Multiple membrane-cytoplasmic domain contacts in the cystic fibrosis transmembrane conductance regulator (CFTR) mediate regulation of channel gating

The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) ion channel mutated in patients with cystic fibrosis. The most common mutation, deletion of phenylalanine 508 (DeltaF508) and many other disease-associated mutations occur in the nucleotide binding domains (NBD) and the cytoplasmic loops (CL) of the membrane-spanning domains (MSD). A recently constructed computational model of the CFTR three-dimensional structure, supported by experimental data (Serohijos, A. W., Hegedus, T., Aleksandrov, A. A., He, L., Cui, L., Dokholyan, N. V., and Riordan, J. R. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 3256-3261) revealed that several of these mutations including DeltaF508 disrupted interfaces between these domains. Here we have used cysteine cross-linking experiments to verify all NBD/CL interfaces predicted by the structural model and observed that their cross-linking has a variety of different effects on channel gating. The interdomain contacts comprise aromatic clusters important for stabilization of the interfaces and also involve the Q-loops and X-loops that are in close proximity to the ATP binding sites. Cross-linking of all domain-swapping contacts between NBDs and MSD cytoplasmic loops in opposite halves of the protein rapidly and reversibly arrest single channel gating while those in the same halves have lesser impact. These results reinforce the idea that mediation of regulatory signals between cytoplasmic- and membrane-integrated domains of the CFTR channel apparently relies on an array of precise but highly dynamic interdomain structural joints.