Heterogeneity of proteoglycans extracted before and after collagenase treatment of human articular cartilage: I. Physical properties related to age

Proteoglycans were isolated from young and mature human articular cartilage 4 different ways: by direct extraction with 4M guanidine hydrochloride (GuHCl); after digestion of the residue from this first extraction with collagenase, by extraction with 4M GuHCl; associatively with 0.5M GuHCl after digestion of the cartilage with collagenase; and dissociatively with 4M GuHCl after digestion of the cartilage with collagenase. The structural properties of these proteoglycans were compared. Proteoglycan aggregates and monomers isolated from second extractions and from young cartilage were of larger hydrodynamic size than proteoglycans isolated from first extractions and mature cartilage, respectively. The same applied to the chondroitin sulfate chain lengths of these proteoglycans. The proteoglycan fraction from second extractions of cartilage contained a larger proportion of monomers than the fraction from first extractions. Associative extraction of mature collagenase-digested cartilage yielded mainly proteoglycan monomers, whereas an appreciable amount of proteoglycan aggregate was also liberated from young collagenase-digested cartilage. Our results indicate that, because of their larger size, proteoglycans from second extractions of cartilage are more entrapped in the collagen network. These large proteoglycans can only be liberated from the matrix after extraction of the smaller proteoglycans, followed by digestion of the residue with collagenase. This indicates that proteoglycans overlap and entangle with the collagen and protect it from degradation by collagenase.     

Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins

The mdr1-type P-glycoproteins (P-gps) confer multidrug resistance to cancer cells by active extrusion of a wide range of drugs from the cell. To study their physiological roles, we have generated mice genetically deficient in the mdr1b gene [mdr1b (−/−) mice] and in both the mdr1a and mdr1b genes [mdr1a/1b (−/−) mice]. In spite of the host of functions speculatively attributed to the mdr1-type P-gps, we found no physiological abnormalities in either strain. Viability, fertility, and a range of histological, hematological, serum–chemical, and immunological parameters were not abnormal in mdr1a/1b (−/−) mice. The high level of mdr1b P-gp normally present in the pregnant uterus did not protect fetuses from a drug (digoxin) in the bloodstream of the mother, although the protein did reduce drug accumulation in the adrenal gland and ovaries. Pharmacologically, mdr1a/1b (−/−) mice behaved similarly to the previously analyzed mdr1a (−/−) mice, displaying, for instance, increased brain penetration and reduced elimination of digoxin. However, both mdr1a and mdr1b P-gps contributed to the extrusion of rhodamine from hematopoietic progenitor cells, suggesting a potential role for the endogenous mdr1-type P-gps in protection of bone marrow against cytotoxic anticancer drugs. This, and the normal viability of mdr1a/1b (−/−) mice, has implications for the use of P-gp-blocking agents in cancer and other chemotherapy. mdr1a/1b (−/−) mice should provide a useful model system to further test the pharmacological roles of the drug-transporting P-gps and to analyze the specificity and effectivity of P-gp-blocking drugs.     

Metabolic and secretory properties of peripheral and synovial granulocytes in rheumatoid arthritis

The metabolic and secretory properties of peripheral and synovial granulocytes of patients with rheumatoid arthritis were investigated with serum- or immunoglobulin-treated zymosan as activators of cell metabolism. During isolation of the synovial cells precautions were taken to prevent in vitro phagocytosis of immune materials present in the synovial fluids. Oxygen uptake, extracellular release of lysosomal enzymes under resting and activated conditions, yield of the isolated granulocytes, and the granule enzyme content of peripheral and synovial cells did not differ significantly from those of peripheral granulocytes from healthy volunteers. In agreement with the biochemical results, intracellular inclusions could be detected in only a few synovial cells with a direct immunofluorescence technique. The possibility that formation of “ragocytes” may be an in vitro phenomenon is discussed.     

Tritiated imipramine binding sites in affective disorders and schizophrenia. Influence of circannual variation

We have investigated platelet [3H]imipramine binding in normal controls and patients with primary endogenous depression (unipolar and bipolar) or schizophrenia. Absolute Bmax values did not differ between subgroups. However, when circannual variation of binding was taken into account by expressing results as percentages of normalised values derived from the multiple linear regression of Bmax values as a function of time of sampling, schizophrenic patients were found to have higher Bmax than normal controls. There was no significant difference between depressed patients and controls, nor between patients exhibiting plasma cortisol suppression and non-suppression after the dexamethasone suppression test. A significant negative correlation was found between relative Bmax values and cerebrospinal 5-HIAA levels in depressed patients.     

DNA Diagnostics of Hereditary Hearing Loss: A Targeted Resequencing Approach Combined with a Mutation Classification System

Although there are nearly 100 different causative genes identified for nonsyndromic hearing loss (NSHL), Sanger sequencing‐based DNA diagnostics usually only analyses three, namely, GJB2, SLC26A4, and OTOF. As this is seen as inadequate, there is a need for high‐throughput diagnostic methods to detect disease‐causing variations, including single‐nucleotide variations (SNVs), insertions/deletions (Indels), and copy‐number variations (CNVs). In this study, a targeted resequencing panel for hearing loss was developed including 79 genes for NSHL and selected forms of syndromic hearing loss. One‐hundred thirty one presumed autosomal‐recessive NSHL (arNSHL) patients of Western‐European ethnicity were analyzed for SNVs, Indels, and CNVs. In addition, we established a straightforward variant classification system to deal with the large number of variants encountered. We estimate that combining prescreening of GJB2 with our panel leads to a diagnosis in 25%–30% of patients. Our data show that after GJB2, the most commonly mutated genes in a Western‐European population are TMC1, MYO15A, and MYO7A (3.1%). CNV analysis resulted in the identification of causative variants in two patients in OTOA and STRC. One of the major challenges for diagnostic gene panels is assigning pathogenicity for variants. A collaborative database collecting all identified variants from multiple centers could be a valuable resource for hearing loss diagnostics.     

Use of a curved-array transducer to reduce interobserver variation in sonographic measurement of thyroid volume in healthy adults

PURPOSE: Sonographic calculation of thyroid volume is used in the diagnosis and follow-up of thyroid diseases. Since the calculated volume of thyroid lobes is highly influenced by the longest (ie, craniocaudal) diameter, we examined whether using a curved-array transducer as opposed to a linear-array transducer to measure the craniocaudal diameter would reduce interobserver variation. METHODS: Three sonographers with different levels of expertise each used a 5-12-MHz linear-array transducer and a 2-5-MHz curved-array transducer to measure the craniocaudal diameter of both thyroid lobes of 25 healthy volunteers. On the basis of these measurements, thyroid lobe volumes were calculated. Single-factor analysis of variance was used to evaluate the interobserver variations between the measurements made by all 3 observers as well as between measurements taken by pairs of observers. A p value of less than 0.05 was considered significant. RESULTS: Using the linear-array transducer to measure the craniocaudal diameter resulted in significant interobserver variation in thyroid volume calculation (p = 0.02), whereas using the convex-array transducer did not. Using either transducer resulted in a highly significant interobserver variation in measurements of the craniocaudal diameter, although the variation was far more pronounced for measurements made with the linear-array transducer (p = 0.0005) than for those made with the curved-array transducer (p = 0.04). For both transducers, the interobserver variations were most pronounced between the most and the least experienced sonographers. CONCLUSIONS: To avoid significant interobserver variation in calculating thyroid lobe volume, we recommend using a curved-array transducer to measure the craniocaudal diameter of the thyroid lobes.