Restriction endonuclease resistant chromatin in human chromosomes

Summary The recent addition of restriction endonucleases in obtaining selective bands in the human genome has added a new dimension to molecular genetics. However, a considerable discrepancy exists in banding patterns produced by AluI in chromosomes 19 and 20, by MboI in chromosomes 4, 5, 8, 21 and 22 and by RsaI in chromosomes 12, 21 and 22. The principal causes of these differences are highlighted.     

Calmodulin supports the force-generating function in desensitized muscle fibers

Externally added calmodulin (CaM) restored Ca2+ regulation for the tension development by skeletal muscle fibers of hamster and rabbit desensitized by the troponin C (TnC) extraction treatment. CaM produced this action by combining with the TnC-denuded sites in the fiber. However, the binding properties differed strikingly from TnC: unlike TnC, CaM binding required the continued presence of Ca2+ and the bound portion was completely released with EGTA in the physiological milieu. The maximal uptake was 1.7 g of CaM/kg of muscle in the present study. The apparent Ca2+ sensitivity for force development with 200 micrograms/ml CaM in the solution was lower than in the native fiber or in the TnC-loaded fiber. The apparent association constant for CaM binding to the TnC-denuded sites was found as 4.9 x 10(5) M-1, and the extrapolated maximum force (Fmax) with CaM was close to PO. The intrinsic CaM level in intact muscle was also measured and was 18.6 mg/kg, amounting to about 1% of the total TnC or the CaM uptake by TnC-denuded fibers. The intrinsic CaM was not dislodged by EDTA treatment, indicating tight binding and suggesting that it exists in a separate pool from the vacated TnC sites adsorbing externally added CaM. The stringent Ca+ dependence of the CaM adsorption to TnC sites in the regulatory complex in the fiber supports the view that the evolutionary replacement of residues in the amino terminus helix portion of the "EF-hand" motif of site IV may be critical for the functional specialization by TnC.     

Lung contains an inhibitor for nicotinatemononucleotide pyrophosphorylase (carboxylating) of NAD biosynthesis

Rat, cow and foal lung extracts contained an inhibitor for the liver NAD biosynthetic-pathway enzyme, nicotinatemononucleotide pyrophosphorylase (carboxylating) [EC 2.4.2.19]. The inhibitor was not dialyzable, was labile at 100 degrees C, was retained by a 30,000 dalton pore size Amicon membrane and, when partially purified by precipitation at 40-100% ammonium sulfate, inhibited the enzyme stoichiometrically. Lung reportedly does not contain nicotinate-mononucleotide pyrophosphorylase or make NAD de novo. However, the inhibitor would mask detection of the enzyme in lung extracts. We detected a low nicotinatemononucleotide pyrophosphorylase-like activity (0.003 +/- 0.001 nanomoles CO2 produced from quinolinic acid per mg of extract protein) in rat lung but none in foal or cow lung.     

Clastogenic effect of pesticides in peripheral lymphocytes of cotton-field workers.

We studied clastogenic effects in peripheral lymphocytes of cotton-field workers who were exposed to different pesticides. All the cells were grown in RPMI 1640 medium for 48 and 72 h. The type of aberrations observed in the exposed group are gaps, breaks, dicentrics, exchanges, rings and polyploidy. The frequency of total chromosomal aberrations increased significantly in male pesticide applicators when compared to controls. A significant decrease in mitotic index was observed in the exposed group as compared to the control group. The 48-h cultures showed high incidence of chromosomal aberrations and low mitotic index when compared to 72-h cultures. The difference in chromosomal aberrations between 48- and 72-h cultures was not significant. 24 out of 26 individuals showed ill health effects such as severe giddiness and nervous disorders.     

Dissociative extraction and partial purification of osteogenin, a bone inductive protein, from rat tooth matrix by heparin affinity chromatography

Implantation of demineralized tooth matrix in subcutaneous sites results in new bone formation locally. The osteoinductive activity of the tooth matrix was dissociatively extracted in 4.0 M guanidine hydrochloride and the residue was devoid of biologic activity. The bone inductive protein, osteogenin, was partially purified by heparin affinity chromatography. The heparin binding fraction initiated the bone differentiation cascade when implanted with guanidine extracted, inactive bone or tooth matrices. These results imply a cooperative interaction between the soluble osteogenin and collagenous substratum in bone induction.     

Metabolism of glomerular basement membrane in short- and long-term streptozotocin diabetic rats

The synthesis of glomerular basement membrane (GBM) total protein and collagen was assessed by two methods in vivo in normal and streptozotocin diabetic rats 4-6 weeks and 42-44 weeks after onset of hyperglycaemia, using L-[2, 3, 3H] proline as a radioactive precursor. The incorporation of tritiated proline into GBM hydroxyproline was used as a measure of collagen synthesis and that into proline as total protein synthesis. The basement membrane fractions from both short- and long-term diabetic rats attained much higher proline and hydroxyproline specific activities compared to normal GBM proline and hydroxyproline specific activities. Early insulin therapy with normalization of blood sugar levels in short-term (4-6 weeks) diabetic rats returned the abnormal increases in GBM total protein and collagen synthesis to normal. By contrast, poor glycaemic control with insulin did not prevent the increases in GBM protein synthesis. The results of the present study suggest that overall enhancement of GBM protein synthesis occurs in both short- and long-term streptozotocin diabetes. Early insulin therapy with normalization of blood sugar levels prevents this increase in GBM protein synthesis. Poor glycaemic control had no effect on abnormal GBM protein synthesis. This may be of potential significance in view of preventing chronic diabetic microvascular complications such as nephropathy.     

Precise location of breakpoints in a frequent reciprocal translocation between chromosomes 11 and 22

One of the most frequent chromosomal translocations in human beings is 11q/22q, which results in the "partial trisomy of 22q syndrome." However, the breakpoint on the long arms of chromosomes 11 and 22 is still a matter of controversy. In the present study, we have used chromosomes from lymphocytes of a neonate who happened to have this classical abnormality, and by R-banding prometaphase chromosomes with acridine orange it has been possible to establish that the translocation between chromosomes 11 and 22 resulted from 3:1 meiotic maternal nondisjunction. A detailed analysis of the chromosome regions involved in this translocation revealed that the breakpoints on chromosomes 11 and 22 were at 11q23.3 and 22q11.1, respectively.     

Autonomy of the wingless mutation in Drosophila melanogaster

Summary T(Y;2) translocations were used to cytologically localise the wingless locus of Drosophila melanogaster. We found that an existing T(Y;2), which is an insertion of a segment of 2L into the Y chromosome, has wg ⁺ within this insert. This Y chromosome was used to generate an attached XY chromosome containing wg ⁺. The mutation claret-nondisjunctional (ca ^nd) was used to induce the loss of this XY chromosome and thus generate gynandromorphs with wg ¹/wg ¹ male tissue and wg ⁺/wg ¹/wg ¹ female tissue. Analysis of these gynanders demonstrated that a genotypically wingless mutant hemithorax is usually also phenotypically mutant in these half body mosaics; thus wg ¹ is discautonomous. This observation is of interest as it is known that wg is not cell autonomous.     

Comparison of AMP and NADH binding to glycogen phosphorylase b

The binding sites for the allosteric activator, AMP, to glycogen phosphorylase b are described in detail utilizing the more precise knowledge of the native structure obtained from crystallographic restrained least-squares refinement than has hitherto been available. Localized conformational changes are seen at the allosteric effector site that include shifts of between 1 and 2 A for residues Tyr75 and Arg309 and very small shifts for the region of residues 42 to 44 from the symmetry-related subunit. Kinetic studies demonstrate that NADH inhibits the AMP activation of glycogen phosphorylase b. Crystallographic binding studies at 3.5 A resolution show that NADH binds to the same sites on the enzyme as AMP, i.e. the allosteric effector site N, which is close to the subunit-subunit interface, and the nucleoside inhibitor site I, which is some 12 A from the catalytic site. The conformations of NADH at the two sites are different but both conformations are "folded" so that the nicotinamide ring is close (approx. 6 A) to the adenine ring. These conformations are compared with those suggested from solution studies and with the extended conformations observed in the single crystal structure of NAD+ and for NAD bound to dehydrogenases. Possible mechanisms for NADH inhibition of phosphorylase activation are discussed.     

Immunohistochemical localization of bursin in epithelial cells of the avian bursa of Fabricius

Antibodies to the avian B-cell-differentiating hormone bursin (lysyl-histidyl-glycine amide) were raised in mice and rabbits by immunizing with bursin conjugates in Freund's adjuvant. Immunohistochemical staining with these bursin-specific antibodies was restricted to follicular and dendritic reticular epithelial cells of the bursa of Fabricius, and was not found in control avian tissues.