The cellular interactions of laminin fragments. Cell adhesion correlates with two fragment-specific high affinity binding sites

The molecular interactions of laminin with several tumor cell lines and skin fibroblasts were investigated by radioligand binding studies and cell attachment assays using laminin, the laminin-nidogen complex, and laminin fragments as substrates and also domain-specific antibodies as inhibitors of cell attachment. The majority of cells showed a dual binding pattern for fragments 1 and 8 which originate from short-arm or long-arm structures of laminin, respectively. Both of these fragments in solution bind to suspended cells with high affinity (KD = 1-10 nM), with the receptor numbers for each fragment depending on the cell type. Competition studies and independent variation of receptor numbers demonstrated that the cell-binding structures on each fragment are different, implicating the existence of two distinct cellular receptors for laminin. The ability of these fragments to act as substrates for cell adhesion correlated with the presence of high affinity binding sites on the cells. However, only antibodies to fragment 8 were able to block cell adhesion to laminin, despite the presence of binding sites for fragment 1. A few cells had very low numbers of high affinity receptors for either fragment 1 or 8. The latter cell type was used to demonstrate that complex formation between laminin and nidogen, which binds to fragment 1 structures, reduces the potential of laminin for cell binding.     

Solid-phase synthesis and side reactions of oligonucleotides containing O-alkylthymine residues

As part of our studies on the molecular mechanism of mutation [Chambers, R. W. (1982) in Molecular and Cellular Mechanisms of Mutagenesis (Lemontt, J. F., & Generoso, W. M., Eds.) pp 121-145, Plenum, New York and London], we wanted to prepare specific oligonucleotides carrying O2- or O4-alkylthymidine residues. Since O-alkylthymine moieties are known to be alkali labile, side reactions were expected during the deprotection procedures used for synthesis of oligonucleotides on a solid support by the classical phosphoramidite method. We have studied these side reactions in detail. Kinetic data show the deprotection procedures displace most O-alkyl groups at rates that make these procedures inappropriate for synthesis of most oligonucleotides carrying O-alkylthymine moieties. We describe alternative deprotection procedures, using readily accessible reagents, that we have used successfully to synthesize a series of oligonucleotides carrying several different O-alkylthymine moieties. The oligonucleotides synthesized are d(A-A-A-A-G-T-alkT-T-A-A-A-A-C-A-T), where alk = O2-methyl, O2-isopropyl, O4-methyl, O4-isopropyl, and O4-n-butyl. This work extends the previously described procedure for the chemical synthesis of oligonucleotides carrying an O4-methylthymine moiety [Li, B. F., Reese, C. B., & Swann, P. F. (1987) Biochemistry 26, 1086-1093] and reports the first chemical synthesis of an oligonucleotide carrying an O2-alkylthymine. The oligonucleotides synthesized have a sequence corresponding to the minus strand that is complementary to the viral strand at the start of gene G in bacteriophage phi X174 replicative form DNA where the normal third codon has been replaced with the ocher codon, TAA.     

Inactivation of dopamine beta-hydroxylase by beta-ethynyltyramine: kinetic characterization and covalent modification of an active site peptide

beta-Ethynyltyramine has been shown to be a potent, mechanism-based inhibitor of dopamine beta-hydroxylase (DBH). This is evidenced by pseudo-first-order, time-dependent inactivation of enzyme, a dependence of inactivation on the presence of ascorbate and oxygen cosubstrates, the ability of tyramine (substrate) and 1-(3,5-difluoro-4-hydroxybenzyl)imidazole-2-thione (competitive multisubstrate inhibitor) to protect against inactivation, and a high affinity of beta-ethynyltyramine for enzyme. Inactivation of DBH by beta-ethynyltyramine is accompanied by stoichiometric, covalent modification of the enzyme. Analysis of the tryptic map following inactivation by [3H]-beta-ethynyltyramine reveals that the radiolabel is associated with a single, 25 amino acid peptide. The sequence of the modified peptide is shown to be Cys-Thr-Gln-Leu-Ala-Leu-Pro-Ala-Ser-Gly-Ile-His-Ile-Phe-Ala-Ser-Gln-Leu- His*- Thr-His-Leu-Thr-Gly-Arg, where His* corresponds to a covalently modified histidine residue. In studies using the separated enantiomers of beta-ethynyltyramine, we have found the R enantiomer to be a reversible, competitive inhibitor versus tyramine substrate with a Ki of 7.9 +/- 0.3 microM. The S enantiomer, while also being a competitive inhibitor (Ki = 33.9 +/- 1.4 microM), is hydroxylated by DBH to give the expected beta-ethynyloctopamine product and also efficiently inactivates the enzyme [kinact(app) = 0.18 +/- 0.02 min-1; KI(app) = 57 +/- 8 microM]. The partition ratio for this process is very low and has been estimated to be about 2.5. This establishes an approximate value for kcat of 0.45 min(-1) and reveals that (S)-beta-ethynyltyramine undergoes a slow turnover relative to that of tyramine (kcat approximately 50 s(-1), despite the nearly 100-fold higher affinity of the inactivator for enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measuring readmission rates.

OBJECTIVE--To assess the feasibility of extracting data on readmissions and readmission rates from Körner data for use as health service indicators. DESIGN--Retrospective analysis of inpatient Körner data for January 1988 to April 1989. SETTING--Three districts in North East Thames region. MAIN OUTCOME MEASURES--Number of readmissions after index discharge for all acute specialties combined and by specialty (general medicine, general surgery, gynaecology, trauma and orthopaedics, and geriatrics); readmission rates at 28 days after index discharge; and rates standardised for age group and sex by specialty and by consultant. RESULTS--All specialties showed an early peak in number of admissions, which levelled off by 28 days. Readmission rates at 28 days were appreciably lower in surgical specialties than in medical specialties (for example, general surgery 4.1% v geriatric medicine 15.1%). They were related to age and sex of the patient. Rates standardised for these variables did not significantly differ by district. Likewise, significant differences in standardised rates were not obtained for consultants within a specialty in one district. CONCLUSIONS--Readmission rates may be measured with Körner data. The pattern of readmissions with time means that readmission rates should be measured at not more than 28 days after the index discharge; the rates require standardisation for age and sex. Annual comparisons of standardised rates may be made among districts for combinations of specialties; those among individual consultants or specialties are unlikely to be statistically valid.     

The pMTL nic- cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing

A series of nic- cloning vectors have been constructed analogous to the pUC plasmids but which are smaller in size and carry more extensive polylinker regions within the lacZ' gene. The vectors pMTL20 and pMTL21 carry six additional sites (AatII, MluI, NcoI, BglII, XhoI and StuI) to those present in pUC18 and pUC19, while pMTL22 and -23 possess eleven new cloning sites (ActII, MluI, NcoI, BglII, XhoI, StuI, NaeI, EcoRV, ClaI, NdeI and NruI). More importantly, the relative order of the restriction sites within the polylinker of these latter vectors has been totally rearranged, relative to pUC18 and pUC19, to facilitate the conversion of DNA fragments with incompatible ends to fragments with compatible termini. The availability of such DNA fragments is a crucial requirement when M13 templates are generated for dideoxy sequencing by the sonication procedure. Derivatives of these vectors have also been constructed which demonstrate improved segregational stability by incorporation of the pSC101 par locus. During the construction of these new vectors data were obtained which demonstrated that the pUC and pMTL plasmids contain a previously unreported single base pair difference within the RNA I/RNA II region (compared to pBR322) responsible for a three-fold increase in plasmid copy number. The pUC and pMTL plasmids were also shown to be functionally nic-, thus affording the lowest categorisation in genetic manipulation experiments.     

Optimisation of total urinary aldosterone estimation: comparison with other laboratory methods for assessment of mineralocorticoid status

We have demonstrated that conventional methods for measuring total urinary aldosterone (TUA) may markedly and inconsistently underestimate aldosterone output, since under the conditions usually employed (pH 1.0), the hydrolysis of aldosterone conjugates in urine is incomplete. The use of more acidic hydrolysis conditions (pH 0.2) overcomes this problem. However free aldosterone may be damaged at this pH. Therefore to accurately measure TUA output, it is necessary to isolate the undamaged aldosterone chromatographically and to correct for procedural losses based on the recovery of aldosterone tracer added to the urine prior to hydrolysis. We compared a number of laboratory estimates of aldosterone status (including urinary free aldosterone) with the 24-h urinary sodium output in normal subjects, since this provides a good bioassay of aldosterone. Sodium output correlated best with "optimised" 24 h TUA, i.e. hydrolysed at pH 0.2, (r = -0.589, P less than 0.001), and with plasma aldosterone (r = -0.504, P less than 0.005). Both aldosterone in random urine specimens and plasma renin activity correlated poorly with 24-h sodium output. Therefore, while the measurement of optimised TUA excretion provides the best index of aldosterone activity, assay of aldosterone in random specimens of plasma, which is more convenient for patient and laboratory, may be adequate for many clinical purposes.     

Underfeeding and exposure to short photoperiod alters rat pineal and Harderian gland lysosomal enzyme activities

Harderian gland (HG) weight and lysosomal enzyme activity were evaluated after 21-day-old female rats were singly caged in a long (LP; 14:10 LD) or short (SP; 8:16 LD) photoperiod and fed on one of two dietary regimens (fed ad libitum or 50% underfed) for 50 days; an additional fed and an underfed group of animals in LP were injected every afternoon with 100 micrograms melatonin. Absolute HG weights were significantly lower in all underfed groups compared to their respective fed controls or to the LP fed control group. Absolute HG weights of underfed rats in SP were significantly lower than the underfed rats in LP. Relative HG weights (mg/100 g body wt) were significantly higher in the underfed saline or melatonin-treated groups compared to their respective fed controls; however, HG of the underfed SP group were not different from SP-fed controls. No significant differences in HG acid phosphatase, hexosaminidase, and beta-glucuronidase activities were observed in any of the treatment groups maintained in LP. Acid phosphatase, hexosaminidase, and beta-glucuronidase activities were significantly elevated in HG of underfed animals maintained in SP compared to their respective fed controls or to the LP-underfed group. Both the underfed control and the underfed-melatonin treated groups had lower pineal protein values than their respective fed groups; underfed animals in 8:16 LD had similar pineal protein values compared to those of the fed control group in SP. Significant effects of photoperiod and underfeeding with no interaction between these variables were observed on pineal acid phosphatase. The fed group maintained in 8:16 LD had significantly higher acid phosphatase activity than the fed group kept in 14:10 LD. In conclusion, underfeeding resulted in severely reduced body weights and absolute Harderian gland weights. Increased activity in certain lysosomal enzymes occurred in both the pineal and Harderian gland and in some instances this was dependent upon the light cycle and dietary regimen to which the animals were exposed.