The hypervariable DXS255 locus contains a LINE-1 repetitive element with a CpG island that is extensively methylated only on the active X chromosome.

The DXS255 locus at Xp11.22 is highly polymorphic due to a 26-bp variable number of tandem repeats (VNTR) motif. In previous studies, one of the MspI sites flanking the VNTR manifested a correlation between methylation and X chromosome inactivation. Here we show, by DNA sequence analysis, that this MspI site is located within the CpG island at the 5' end of a LINE-1 element, which is 2.5 kb from the VNTR. The methylation status of the CpG island was assessed in Southern blotting experiments using the methylation-sensitive enzymes HpaII, HhaI, and BssHII. All these sites were completely methylated on active X chromosomes, consistent with previously reported findings of full methylation of LINE-1 elements throughout the genome. However, on inactive X chromosomes these sites were predominantly unmethylated, although patterns were found to be heterogeneous. The results suggest that LINE-1 elements on the inactive X chromosome are not suppressed by full methylation of their CpG islands. The differential methylation of the DXS255 CpG island provides the basis for a highly informative X inactivation analysis system.     

Ultrastructural analysis of major basement membrane types in rhesus monkey Macaca mulatta acellular renal cortex

Increasing interest in animal models of human nephropathies have led to a number of renal studies in nonhuman primates. In the current investigation, sequential detergent extraction of cellular elements was carried out on renal cortical tissue blocks from rhesus monkey in an effort to demonstrate clearly the morphological features of major basement membrane (BM) types and their associated extracellular matrix (ECM). LM and TEM views of acellular tissue blocks demonstrate planar arrangements of ECM components, while SEM studies provide striking three-dimensional images of their surface characteristics. All major BM types maintain their in vivo histoarchitectures despite the absence of cells. We propose that the intrinsic structural rigidity of tubular (TBM), Bowman's capsule (BCBM) and peritubular capillary BM (PTCBM) may be related to to their close external association with collagenous fibrils, while glomerular BM (GBM) may be internally supported by a network of mesangial matrix (MM) plates and trabeculae which extend onto internal surfaces of peripheral GBM loops. Thicknesses of rhesus monkey renal BMs show that they are similar to those seen in the laboratory rat and, in general, BCBM greater than TBM greater than GBM greater than PTCBM. We conclude that rhesus monkey renal BMs closely resemble those described by us in the human [J. Ultrastruct. Res. 82: 96-110, 1983] and that this species offers an attractive model for studies of renal diseases of BM origin-notably diabetes mellitus.     

Changes in prolactin in peripheral plasma during lactation in the brushtail possum Trichosurus vulpecula

A heterologous double-antibody radioimmunoassay has been validated for prolactin in plasma and pituitary preparations of T. vulpecula. Serial dilutions of crude pituitary homogenates and plasmas from several marsupials and purified prolactin from the tammar, Macropus eugenii, showed parallel dose response curves. In both male and female possums plasma prolactin concentrations increased in response to a single intravenous injection of thyrotrophin releasing hormone. Plasma prolactin concentrations were measured in six lactating females (June-November) and in four non-lactating females (July-October). In the following year prolactin levels were also measured in 11 possums with young less than 50 days old and in 24 possums with young aged between 100 and 145 days. In early lactation prolactin concentrations were low (less than 8 ng/ml) but increased to high levels (greater than 30 ng/ml) by 120 days and remained high until about 160 days of lactation. Thereafter concentrations declined although the young continued to take milk from the mother for a further 30-50 days. The changes in plasma prolactin concentrations throughout lactation are very similar to those described for the tammar, and this unusual pattern appears to be common to marsupials. Non-lactating possums showed no consistent changes in plasma prolactin concentrations between July and October.     

The effect of ETH 1001 on ion fluxes across red blood cell membranes

The calcium selective ionophore, ETH 1001, and the divalent cation ionophore, A23187, promoted Ca2+ flux across RBC membranes under various experimental conditions. ETH 1001 did not promote the passive movement of Mg2+ whereas A23187 did. The results confirm the potential application of ETH 1001 as a Ca2+ selective ionophore for biological membranes.     

Cellular gene expression in papillomas of the choroid plexus from transgenic mice that express the simian virus 40 large T antigen.

Transgenic mice that contain the simian virus 40 (SV40) enhancer-promoter and large tumor (T) antigen gene develop papillomas of the choroid plexus. The tumors remain well differentiated on histological examination and express normal levels of tissue-specific mRNAs for transthyretin (TTR) and the 5-HT1C serotonin receptor, two differentiated cell markers. Both Northern (RNA) blot analysis and in situ cytohybridization have been used to monitor the steady-state levels of the mRNAs from the viral oncogene (T antigen) and from several cellular oncogenes. In situ hybridization demonstrated, in serial sections, increased levels of both T antigen mRNA and p53 mRNA localized in the tumor tissue but not in the normal brain tissue. The ratios of the steady-state levels of mRNA for p53/TTR and p53/L32, a ribosomal protein gene, were 2- to 20-fold higher in the tumor tissue than in the normal choroid plexus tissue. Several other oncogenes did not show elevated levels of mRNA in these tumors. p53 protein levels were not detectable in normal brain tissue, but p53 levels were very high in tumor tissue in which all of the p53 was found in a complex with the SV40 large T antigen. These data continue to show a close relationship between SV40 T-antigen-mediated tumorigenesis and the role of p53 in these tumors.     

Sequential1H-NMR assignments of neurotoxin III from the sea anemoneHeteractis macrodactylus and structural comparison with related toxins

The complete sequence-specific assignment of resonances in the¹H-NMR spectrum of the polypeptide neurotoxin III (Hm III) from the sea anemoneHeteractis macrodactylus is described. Comparison of the chemical shifts and pattern of NOEs for Hm III with those for the related toxin Hp III fromHeteractis paumotensis, which differs only in the substitution of Asn for Tyr at position 11, shows that the overall secondary and tertiary structures are conserved. The largest differences in chemical shift caused by the substitution at position 11 are observed for the NH resonances of Arg-13, Thr-14, Ala-15, Leu-17, and Cys-26. The CH resonances influenced most are those of ASP-6, Gly-9, Leu-17, and Glu-42, while the most affected CH resonances are from Leu-17, Glu-28, and Lys-32. The absence of long-range NOEs to the aromatic ring of Tyr-11 as well as the lack of significant chemical shift effects on residues outside the loop comprising residues 7–16 confirm that this part of the loop makes no long-lived contacts with the rest of the molecule. The deviations from random coil shifts of Hm III are compared with those of the related anemone toxins Hp II, Hp III, and toxin I fromStichodactyla helianthus (Sh I). The similarity in deviations in chemical shift as a function of sequence position for these four toxins emphasizes the overall structural homology among these polypeptides.     

Solution properties ofEscherichia coli-expressed VH domain of anti-neuraminidase antibody NC41

The V_H domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAG^TM), has been expressed in high yield (15–27 mg/L) inEscherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The V_H-FLAG was composed of three isoforms (pI values of 4.6, 4.9, and 5.3) and the V_H molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the V_H isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20°C and concentrations of 5–10mg/ml the V_H domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its¹H NMR spectrum. Reagents such as CHAPS,n-octylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the V_H molecule, prevented dimerization of the V_H and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.