A new biallelic DNA polymorphism of the human COL5A1 gene

A cDNA probe of the human COL5A1 gene detects a frequent biallelic PstI polymorphism. Allele A has a frequency of 54% whereas that of allele B is 46%. This restriction fragment length polymorphism provides a useful marker for linkage analysis in 9q34.3.     

Production of amylase(s) by Schwanniomyces castellii and Endomycopsis fibuligera

Schwanniomyces castellii and Endomycopsis fibuligera Produced extracellular amylase(s) when grown on various carbon sources and at different pH values. Both yeast species showed significant amylase synthesis in the presence of either maltose or soluble starch. On the other substrates tested (glucose, cellobiose, sucrose, trehalose, melezitose, raffinose, ethanol, glycerol) differences were found regarding growth and amylase production. Free glucose in the culture medium apparently inhibited enzyme synthesis. The pH range allowing maximal growth and amylase production was 4.5–6.0 for E. fibuligera and 5.5–7.0 for S. castellii.     

Transverse sarcomere splitting. A possible means of longitudinal growth in crab muscles

Transversely split sarcomeres are seen in mouthpart muscles of the blue crab in the electron microscope. Sarcomeres split only at the H zone. Two new sarcomeres are formed by a Z disk which appears in the H zone of the splitting sarcomere. Splitting may involve breaking of the thick filaments in the H zone, elongation of these filaments, and formation of both new actin filaments and Z-disk materials, Sarcomere splitting would allow longitudinal growth of muscle cells without lengthening of sarcomeres and concomitant changes in contractile properties.     

The pharmacokinetics of tritiated ethinylestradiol in the rabbit

The disappearance of ethinylestradiol from the blood of rabbits has been studied, following the intravenous administration of this steroid. The disappearance followed two exponentials, the first having a half life ($\text{t}\text{1}\text{2}$) of 5.5 min and the second, apparently terminal exponential was also rapid ($\text{t}\text{1}\text{2}\text{= 69}\text{min}$). The plasma clearance was 150 ml/min which suggests almost total clearance of this steroid during a single passage through the liver. Bile contained a significant concentration of EE conjugates and thus this steroid could undergo enterohepatic recirculations. A large oral dose of unlabelled EE, given prior to intravenous administra tion of tritiated EE, considerably altered the pharmacokinetics of the latter by saturating both phase one metabolism (changes of the steroid nucleus) and the secretion of conjugates into bile. It was not clear whether phase two metabolism (conjugation) was also saturated.     

Secretin modulation of behavioral and physiological functions in the rat

The effect of secretin on behavioral and physiological functions in the rat was investigated. Secretin injected intracerebroventricularly (ICV) significantly increased defecation and decreased novel-object approaches in rats. The peptide showed no significant effects on stereotypic behavior (gnawing, grooming and rearing), open-field locomotor activity however was significantly decreased, an effect that was probably due to a decreased propensity for the rats to initiate locomotor responses. In addition, secretin showed significant effects on respiration rate in anesthetized rats. When the peptide was injected in the lateral ventricle a decrease in respiration rate occurred, but when the brain was perfused from the lateral ventricle to the cisterna magna increases in respiration rate occurred. These data, combined with the facts that secretin and secretin receptors have been identified in the brain indicate that secretin may play a neurotransmitter or neuroregulator role in the central nervous system.     

Structure-function analysis of casein kinase 2 with synthetic peptides and anti-peptide antibodies.

Casein kinase 2 (CK2) is a ubiquitous, multifunctional protein-seryl/threonyl kinase that has been implicated in cellular regulation. Synthetic peptides were patterned after three highly conserved regions in CK2: the N terminus (CK2-NT); the lysine-rich, kinase subdomain III segment (CK2-III) (nomenclature of Hanks et al. (Hanks, S. K., Quinn, A. M., and Hunter, T. (1988) Science 241, 42-52)); and a 10-residue segment located near kinase subdomain X that is shared between CK2 and p34cdc2 (CK2/cdc2). The CK2-III and CK2/cdc2 peptides markedly stimulated the autophosphorylation of the alpha- and alpha'-subunits of purified CK2 from sea star oocytes, and they elicited up to 2-fold increases in its casein or phosvitin phosphotransferase activity. These peptides completely reversed nearly total inhibition of CK2 phosphotransferase activity toward itself, casein, and phosvitin by either heparin or poly(Glu,Tyr; 4:1), whereas CK2-NT was ineffective. Elution of CK2 from heparin-agarose with the CK2-III peptide indicated that this region of CK2 might mediate heparin binding to CK2. Affinity-purified rabbit polyclonal antibodies developed against both CK2-III and CK2/cdc2, but not CK2-NT, also produced up to 1.8-fold enhancements of the casein and phosvitin phosphotransferase activities of purified CK2. All three of the antipeptide antibody preparations immunoreacted with the alpha- and alpha'-subunits of CK2 on Western blots. These studies indicate that kinase subdomains III and X are involved in the modulation of CK2 phosphotransferase activity.     

MK-801 Prevents 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Parkinsonism in Primates

In cynomologus monkeys, systemic administration of MK-801, a noncompetitive antagonist for the N-methyl-D-aspartate receptor, prevented the development of the parkinsonian syndrome induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MK-801 also attenuated dopamine depletion in the caudate and putamen and protected dopaminergic neurons in the substantia nigra from the degeneration induced by the neurotoxin. Nevertheless, 7 days after MPTP administration in the caudate and putamen of monkeys also receiving MK-801, the levels of toxic 1-methyl-4-phenylpyridinium were even higher than those measured in monkeys receiving MPTP alone. This indicates that the protective action of MK-801 is not related to MPTP metabolism and strongly suggests that, in primates, the excitatory amino acids could play a crucial role in the mechanism of the selective neuronal death induced by MPTP.     

The paradoxical nature of DNA damage and cell death induced by 125I decay.

Chinese hamster ovary cells were synchronized at the G1/S-phase boundary of the cell cycle and pulse-labeled for 10 min with 125I-iododeoxyuridine 30 min after entering the S phase. Cell samples were harvested for freezing and 125I-decay accumulation at intervals ranging from 15 to 480 min after termination of labeling. The survival data showed a marked shift from cell killing characteristic of low-LET radiation to that more characteristic of killing by high-LET radiation with increasing intervals between DNA pulse-labeling and decay accumulation. Cells harvested and frozen within 1 h after pulse-labeling yielded a low-LET radiation survival response with a pronounced shoulder and a large D0 of up to 0.9 Gy. With longer chase periods the shoulder and the D0 decreased progressively, and cells harvested 5 h after pulse-labeling or later exhibited a high-LET survival response (D0: 0.13 Gy). Two interpretations for these findings are discussed. (1) If DNA is the sole target for radiation death, the results indicate that DNA maturation increases radiation damage to DNA or reduces damage repair. (2) If radiation cell death involves damage to higher-order structures in the cell nucleus, the findings suggest that newly replicated DNA is not attached to these structures during the initial low-LET period, but 125I starts to induce high-LET radiation effects as labeled DNA segments become associated with the target structure(s). On balance, or data favor the latter interpretation.     

A small protein in model membranes: a time-resolved fluorescence and ESR study on the interaction of M13 coat protein with lipid bilayers

Model membranes with unsaturated lipid chains containing various amounts of M13 coat protein in the -helical form were studied using time-resolved fluorescence and ESR spectroscopy. The lipid-to-protein (L/P) ratios used were > 12 to avoid protein-protein contacts and irreversible aggregation leading to -polymeric coat protein. In the ESR spectra of the 12-SASL probe in dioleoyl phosphatidylcholine (DOPC) bilayers no second protein induced component is observed upon incorporation of M13 coat protein. However, strong effects are detected on the ESR lineshapes upon changing the protein concentration. The ESR lineshapes are simulated by assuming a fixed ratio between the parallel (D) and perpendicular (D) diffusion coefficients of 4, and an order parameter equal to zero. It is found that increasing the protein concentration from L/P to L/P 15 results in a decrease of the rotational diffusion coefficient D from 3.4 × 10⁷ to 1.9 × 10⁷ s^–1. In the time-resolved fluorescence experiments with DPH-propionic acid as a probe, it is observed that increasing the M13 coat protein concentration causes an increase of the two fluorescent lifetimes, indicating an increase in bilayer order. Analysis of the time-resolved fluorescence anisotropy decay allows one to quantitatively determine the order parameters P₂ and P₄, and the rotational diffusion coefficient D of the fluorescent probe. The order parameters P₂ and P₄ increase from 0.34 to 0.55 and from 0.59 to 0.77, respectively, upon adding M13 coat protein to DOPC bilayers with an L/P ratio of 35. The rotational diffusion coefficient D of the DPH-propionic acid probe decreases on incorporating M13 coat protein, in accordance with the ESR results. It is concluded that M13 coat protein in the -monomeric state is not able to produce a long living lipid boundary shell and consequently an immobilization of the lipids. An overall effect on the lipids is induced, resulting in a reduction in the dynamics and an increase in average lipid order. The hydrophobic region of M13 coat protein is proposed to perfectly match the lipid bilayer, resulting in a relatively small distortion of the bilayer structure of the lipid system.     

Single-stranded molecules in DNA preparations from cultured mammalian cells at different moments of cell cycle

Long single-stranded DNA molecules have been observed at electron microscope in DNA preparations from synchronized Chinese hamster cells. The amount of single strandedness in parental DNA increases following a prolonged block of DNA synthesis by hydroxyurea as judged by the results obtained using an improved hydroxyapatite chromatography (Hanania et al., 1975). As far as newly replicated DNA is concerned, an increase of the single strand amount has been observed in DNA preparations from cells actively synthesizing DNA.