Simple procedure for assessing relative quantities of neutral and acidic sugars in mucin glycoproteins: its use in assessing cyclical changes in cervical mucins.

A simple histochemical procedure for assessing relative amounts of neutral and acidic sugars in mucin glycoproteins, and its application in the study of cyclical changes of human cervical mucins, is described. This procedure, the saponification/selective periodate oxidation/borohydride reduction/alcian blue pH 2.5/periodic acid Schiff (KOH/PA*/Bh/Ab 2.5/PAS) method, uses a selective oxidation step to remove the PAS positivity of sialic acid; thus only neutral sugars stain positively with PAS, and acidic sugars (O-sulphate esters and carboxyl groups) stain with alcian blue. This differs from the KOH/Ab/PAS technique which stains sialic acid residues with both alcian blue and PAS. Applying the KOH/PA*/Bh/Ab 2.5/PAS technique to the study of cyclical changes of human cervical mucins, a decreased neutral:acidic sugar ratio in the secretory phase mucins compared with those of the proliferative phase was found. This difference was not seen with KOH/Ab/PAS staining in the same cases. The techniques and reagents used in this procedure can be easily applied in a clinical histopathology laboratory.     

Immunoglobulin concentrations in children receiving treatment for acute lymphoblastic leukaemia

As part of a wider survey of infections and defence mechanisms, concentrations of serum immunoglobulins IgG, IgA, and IgM were measured regularly by single radial immunodiffusion in a group of children receiving treatment for acute lymphoblastic leukaemia while in their first remission. IgM concentrations were often markedly raised at diagnosis, and IgG and IgA concentrations both began falling within one month of diagnosis. Four months after diagnosis all three had fallen significantly, but this tendency stopped after six months. Only IgG partially recovered, and non returned to pretreatment concentrations even in children followed for two years. The results suggest that vincristine and prednisone are the major factors that initiated the fall in IgG and IgA. Lowered IgM concentrations may have been due to cranial irradiation or continuous maintenance treatment, or both.     

Glomerulocystic kidney disease in a Belgian Malinois dog: an ultrastructural, immunohistochemical, and lectin-binding study

Renal cysts in the cortex of a juvenile Belgian Malinois dog with acute renal failure were studied by means of light, scanning and transmission electron microscopy, immunohistochemistry for intermediate filaments, and binding for wheat germ agglutinin (WGA), peanut agglutinin (PNA), and Maclura pomifera agglutinin (MPA) lectins to determine the morphological and histochemical features of the epithelial cells of these cysts. The cysts were renal corpuscles with expanded urinary space. Glomerular tufts were small with poorly developed capillary loops and increased mesangial matrix. Continuity with the proximal tubule was evident in some cystic glomeruli. Two cell types lined Bowman's capsule. One was squamous with a central cilium and microvilli. The other had morphological and histochemical features of immature podocytes (parietal podocytes). These cells were round and protruded into the urinary space; they had thick cytoplasmic projections that resembled foot processes of podocytes, microvilli, and filtration slits. The parietal podocytes expressed vimentin and cytokeratins and had affinity for WGA as do normal immature podocytes. These features suggest that the parietal podocytes are derived by metaplasia of the parietal cells. The basement membrane of Bowman's capsule was irregularly thickened and showed multifocal glycosylation changes with lectin histochemistry (WGA, PNA, MPA) in areas adjacent to the parietal podocytes. Histologic and ultrastructural findings in this dog are consistent with glomerulocystic kidney disease. This is the second report of canine glomerulocystic kidney disease. Features are similar to those of the human counterpart, but it is unclear whether genetic defects cause the disease in the dog. The presence of parietal podocytes in all cysts suggests that abnormal differentiation may play an important role in the pathogenesis of this type of polycystic kidney disease.     

Decreased lung capillary blood volume post-exercise is compensated by increased membrane diffusing capacity

The diffusing capacity of the lung for carbon monoxide (DLCO) decreases to below the pre-exercise value in the hours following a bout of intense exercise. Two mechanisms have been proposed: (1) development of pulmonary oedema and (2) redistribution of central blood volume to peripheral muscles causing a reduction in pulmonary capillary blood volume (V_c). In the present study DLCO, V_c and the membrane diffusing capacity (D_m) were measured in nine healthy females using a rebreathing method, in contrast to the single breath technique employed in previous studies. DLCO, V_c and D_m were measured before and at 1, 2, 3, 16 and 24 h following maximal treadmill exercise. Compared with pre-exercise values, DLCO was depressed by up to 8.9 (3.0)% (P<0.05) for the first 3 h following exercise, but had returned to pre-exercise values by 16 h post-exercise. V_c fell by 21.2 (4.1)% (P<0.05) at 3 h post-exercise, but at the same time D_m increased by 14.7 (9.1)%. It was concluded that: (1) the increase in D_m made it unlikely that the fall in DLCO was due to interstitial oedema and injury to the blood gas barrier; (2) on the other hand, the reduction in DLCO following exercise was consistent with a redistribution of blood away from the lungs; and (3) the trend for D_m and V_c to reciprocate one another indicates a situation in which a fall in V_c nevertheless promotes gas transfer at the respiratory membrane. It is suggested that this effect is brought about by the reorientation of red blood cells within the pulmonary capillaries following exercise.     

Elevated telomere-telomere recombination in WRN-deficient, telomere dysfunctional cells promotes escape from senescence and engagement of the ALT pathway

Werner Syndrome (WS) is characterized by premature aging, genomic instability, and cancer. The combined impact of WRN helicase deficiency and limiting telomere reserves is central to disease pathogenesis. Here, we report that cells doubly deficient for telomerase and WRN helicase show chromosomal aberrations and elevated recombination rates between telomeres of sister chromatids. Somatic reconstitution of WRN function, but not a WRN helicase-deficient mutant, abolished telomere sister chromatid exchange (T-SCE), indicating that WRN normally represses T-SCEs. Elevated T-SCE was associated with greater immortalization potential and resultant tumors maintained telomeres via the alternative lengthening of telomere (ALT) pathway. We propose that the increased incidence of chromosomal instability and cancer in WS relates in part to aberrant recombinations between sister chromatids at telomeres, which facilitates the activation of ALT and engenders cancer-relevant chromosomal aberrations and tumor formation.     

Delay between pregnancy confirmation and sickle cell and [corrected] thalassaemia screening: a population-based cohort study.

BACKGROUND: Antenatal sickle cell and thalassaemia screening sometimes occurs too late to allow couples a choice regarding termination of affected fetuses. The target gestational age for offering the test in the UK is 10 weeks. AIM: To describe the proportion of women screened before 70 days' (10 weeks') gestation and the delay between pregnancy confirmation in primary care and antenatal sickle cell and thalassaemia screening. DESIGN OF STUDY: Cohort study of reported pregnancies. SETTING: Twenty-five general practices in two UK inner-city primary care trusts offering universal screening. METHOD: Anonymised data on all pregnancies reported to participating general practices was collected for a minimum of 6 months. RESULTS: There were 1441 eligible women intending to proceed with their pregnancies, whose carrier status was not known. The median (interquartile range [IQR]) gestational age at pregnancy confirmation was 7.6 weeks (6.0-10.7 weeks) and 74% presented before 10 weeks. The median gestational age at screening was 15.3 weeks (IQR = 12.6-18.0 weeks), with only 4.4% being screened before 10 weeks. The median delay between pregnancy confirmation and screening was 6.9 weeks (4.7-9.3 weeks) After allowing for practice level variation, there was no association between delay times and maternal age, parity, and ethnic group. CONCLUSION: About 74% of women consulted for pregnancy before 10 weeks' gestation but fewer than 5% of women were screened before the target time of 10 weeks. Reducing the considerable delay between pregnancy confirmation in primary care and antenatal sickle cell and thalassaemia screening requires methods of organising and delivering antenatal care that facilitate earlier screening to be developed and evaluated.     

Recognition surfaces of MHC class I

Summary: Recent crystallographic results have provided dose to atomic resolution views of the recognition events mediated by MHC class I molecules. The specificity-conferring interaction of MHC class I/peptide with a T-cell antigen receptor (TCR) appears dependent on certain key interactions with the MHC scaffold. These interactions, in particular those of the TCR Va domain, define a standard orientation for TCR binding. Previous studies on biologically significant variations in the TCR recognition surface presented by a series of MHC/variant peptide complexes can be reassessed in the light of this TCR-building mode. The interaction of CDS with MHC class I resembles that between antibody and antigen in the use of loops from the CD8 structure. The interaction is of very low affinity and buries equivalent surface area to that between the TCR and MHC class I but while the TCR/MHC interface shows poor surface shape complementarity the match in the conservative interaction between MHC and CDS is precise.     

Identification and characterization of the scl gene encoding a group A Streptococcus extracellular protein virulence factor with similarity to human collagen

Group A Streptococcus (GAS) expresses cell surface proteins that mediate important biological functions such as resistance to phagocytosis, adherence to plasma and extracellular matrix proteins, and degradation of host proteins. An open reading frame encoding a protein of 348 amino acid residues was identified by analysis of the genome sequence available for a serotype M1 strain. The protein has an LPATGE sequence located near the carboxy terminus that matches the consensus sequence (LPXTGX) present in many gram-positive cell wall-anchored molecules. Importantly, the central region of this protein contains 50 contiguous Gly-X-X triplet amino acid motifs characteristic of the structure of human collagen. The structural gene (designated scl for streptococcal collagen-like) was present in all 50 GAS isolates tested, which together express 21 different M protein types and represent the breadth of genomic diversity in the species. DNA sequence analysis of the gene in these 50 isolates found that the number of contiguous Gly-X-X motifs ranged from 14 in serotype M6 isolates to 62 in a serotype M41 organism. M1 and M18 organisms had the identical allele, which indicates very recent horizontal gene transfer. The gene was transcribed abundantly in the logarithmic but not stationary phase of growth, a result consistent with the occurrence of a DNA sequence with substantial homology with a consensus Mga binding site immediately upstream of the scl open reading frame. Two isogenic mutant M1 strains created by nonpolar mutagenesis of the scl structural gene were not attenuated for mouse virulence as assessed by intraperitoneal inoculation. In contrast, the isogenic mutant derivative made from the M1 strain representative of the subclone most frequently causing human infections was significantly less virulent when inoculated subcutaneously into mice. In addition, both isogenic mutant strains had significantly reduced adherence to human A549 epithelial cells grown in culture. These studies identify a new extracellular GAS virulence factor that is widely distributed in the species and participates in adherence to host cells and soft tissue pathology.     

Mutagenicity of some benzidine congeners and their N-acetylated and N,N'-diacetylated derivatives in different strains of Salmonella typhimurium

The Ames Salmonella/microsome test was used to compare the mutagenic response of Salmonella typhimurium TA100, TA98, TA1538, and TA1535 to 12 benzidine derivatives, ie, benzidine, 3,3'-dimethoxybenzidine, 3,3'-dimethylbenzidine, 3,3'-dichlorobenzidine, and the corresponding N- and N,N'-diacetylated derivatives. With a few exceptions, the mutagenic response to this series of compounds varied in the order TA98 greater than TA1538 greater than TA100 greater than TA1535 = 0, and the N-monoacetylated derivatives were more mutagenic than either the parent diamines or the N,N'-diacetyl derivatives. The relative mutagenicities of the parent amines for TA98 were 3,3'-dichlorobenzidine much greater than 3,3'-dimethoxybenzidine greater than benzidine greater than 3,3'-dimethylbenzidine.