Deletion of the Nuclear Localization Sequences and C-Terminus of PTHrP Impairs Embryonic Mammary Development but also Inhibits PTHrP Production

Parathyroid hormone-related protein (PTHrP) can be secreted from cells and interact with its receptor, the Type 1 PTH/PTHrP Receptor (PTHR1) in an autocrine, paracrine or endocrine fashion. PTHrP can also remain inside cells and be transported into the nucleus, where its functions are unclear, although recent experiments suggest that it may broadly regulate cell survival and senescence. Disruption of either the PTHrP or PTHR1 gene results in many abnormalities including a failure of embryonic mammary gland development in mice and in humans. In order to examine the potential functions of nuclear PTHrP in the breast, we examined mammary gland development in PTHrP (1–84) knock-in mice, which express a mutant form of PTHrP that lacks the C-terminus and nuclear localization signals and which can be secreted but cannot enter the nucleus. Interestingly, we found that PTHrP (1–84) knock-in mice had defects in mammary mesenchyme differentiation and mammary duct outgrowth that were nearly identical to those previously described in PTHrP^−/− and PTHR1^−/− mice. However, the mammary buds in PTHrP (1–84) knock-in mice had severe reductions in mutant PTHrP mRNA levels, suggesting that the developmental defects were due to insufficient production of PTHrP by mammary epithelial cells and not loss of PTHrP nuclear function. Examination of the effects of nuclear PTHrP in the mammary gland in vivo will require the development of alternative animal models.     

Transitory hematologic effects of moderate exercise are not influenced by iron supplementation

A young women's exercise/fitness class tested the idea that administration of supplemental iron would prevent "sports anemia" that may develop during exercise and training and improve iron status of exercising females of menstrual age. Fifteen women (aged 18-37) were selected for each of three treatment groups: (1) no supplemental iron; (2) 9 mg X d-1 of Fe; and (3) 18 mg X d-1 of Fe (1 US Recommended Daily Allowance). Women exercised at approximately 85% of maximal heartrate for progressively increasing lengths of time in a jogging program and worked up to 45 min of exercise 4 d X week-1 for 8 weeks. Hematologic analysis was performed in weeks 1, 5, and 8. A significant decline in hemoglobin (Hb) concentration and hematocrit (Hct) was observed at week 5 when all data were examined without regard for iron intake; these red cell indices returned to pre-exercise levels by week 8. Reduction of mean cell hemoglobin concentration (MCHC) indicated that the midpoint decline was not caused by simple hemodilution during exercise. Serum ferritin (SF) concentration changed in parallel with Hb and Hct. Although the midpoint decline in SF was not statistically significant, it ruled out the possibility that turnover of red cell iron was directed to storage. Lowered MCHC and SF suggested lower availability of iron during the synthesis of a new generation of red cells. Few iron treatment effects of magnitude were observed. Iron did not prevent the midpoint decline in Hb concentration. Iron intake did not affect SF, serum iron, transferrin saturation, or final Hb, and Hct.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dihydrofolate reductase: low-resolution mass-spectrometric analysis of an elastase digest as a sequencing tool (Short Communication)

An elastase digest of a protein of unknown structure, dihydrofolate reductase, was studied by mass spectrometry. This soluble digest contained a large number of small peptides in different yields, within the ideal molecular-weight range (200-1200) for mixture-analysis mass spectrometry. Sequences of the major component peptides in the digest are reported.     

Evolution of the ATP-binding-cassette transmembrane transporters of vertebrates

The ATP-binding-cassette transmembrane transporters (ABC transporters) known from vertebrates belong to four major subfamilies: (1) the P- glycoproteins (Pgp); (2) the cystic fibrosis transmembrane conductance regulators (CFTR); (3) the Tap proteins encoded with the major histocompatibility complex of mammals; and (4) the peroxisomal membrane proteins. Both Pgp and CFTR have a structure suggesting a past internal gene duplication; a phylogenetic analysis indicated that these duplications occurred independently, while an independent tandem gene duplication occurred in the case of the Tap family. Both the Pgp and Tap proteins show evidence of relationship to bacterial ABC transporters lacking internal duplication, and both are significantly more closely related to the HlyB and MsbA families of transporters from purple bacteria than they are to ABC transporters from nonpurple bacteria. The simplest hypothesis to explain this observation is that eukaryotic Pgp and Tap genes are descended from a mitochondrial gene or genes that were subsequently translocated to the nuclear genome. The Pgp genes of eukaryotes are characterized by a remarkable degree of convergent evolution between the ATP-binding cassettes of their N- terminal and C-terminal halves, whereas no such convergence is seen between the two halves of CFTR genes or between the duplicated Tap genes. Exon 13 of the CFTR gene, which encodes a putative regulatory domain not found in other ABC transporters apart from CFTR, showed high levels of both synonymous and nonsynonymous difference in comparisons among different mammalian species, suggesting that this region is a mutational hot spot.      

Evidence for the presence of β-lactamase inStreptomyces glaucescens and its inhibition by sodium clavulanate

Streptomyces glaucescens is shown to possess -lactamase activity which is inhibitable by clavulanate. This is important in regard to its use as a cloning host for enzymes of \-lactam biosynthesis.     

Kinetics and mechanism of action of muscle pyruvate kinase

1. The mechanism of rabbit muscle pyruvate kinase was investigated by measurements of fluxes, isotope trapping, steady-state velocity and binding of the substrates. All measurements were made at pH8.5 in Tris/HCl buffer and at 5mm-free Mg(2+). 2. Methods of preparing [(32)P]phosphoenolpyruvate from [(32)P]P(i) in high yield and determining [(32)P]-phosphoenolpyruvate and [8-(14)C]ADP are described. 3. The ratio Flux of ATP to ADP/Flux of ATP to phosphoenolpyruvate (measured at equilibrium) increased hyperbolically with ADP concentration from unity to about 2.1 at 2mm-ADP, but was unaffected by phosphoenolpyruvate concentration. Since the ratio is greater than unity, one pathway for the addition of substrates must involve phosphoenolpyruvate adding first to the enzyme in a rate-limiting step. However, the substrates must also add in the alternative order, because of the non-linear increase in the ratio with ADP concentration and because the rate of increase is very much less than that predicted from the steady-state velocity data for an ordered addition. The lack of influence of phosphoenolpyruvate on the ratio is consistent with the rapid addition of ADP in the alternative pathway. At low ADP concentrations the alternative pathway contributes less than 33% to the total reaction. 4. Isotope trapping was observed with [(32)P]phosphoenolpyruvate, confirming that when phosphoenolpyruvate adds first to the enzyme it is in a rate-limiting step. The release of phosphoenolpyruvate from the ternary complex must also be a slow step. Trapping was not observed with [8-(14)C]ADP, hence the addition of ADP to the free enzyme must be rapid unless its dissociation constant is very large (>20mm). 5. Binding studies showed that 4mol of [(32)P]phosphoenolpyruvate binds to 1mol of the enzyme, probably unligated to Mg(2+), with a dissociation constant appropriate to the mechanism indicated above. Binding of [8-(14)C]ADP could not be detected, and hence the binding of ADP occurs by a low-affinity step. The latter is also demanded by the steady-state velocity data. 6. The ratio Flux of phosphoenolpyruvate to ATP/Flux of phosphoenolpyruvate to pyruvate (determined from the incorporation of label into phosphoenolpyruvate from [3-(14)C]-pyruvate or [gamma-(32)P]ATP during the forward reaction) did not differ significantly from unity. Steady-state velocity data predicted grossly different flux ratios for ordered dissociations of the products, and the results indicate that the dissociation must be rapid and random. The data also exclude a Ping-Pong mechanism. 7. Permissible rate constants for the above mechanism are calculated. The results indicate a high degree of cooperativity in binding, whatever the order of addition of substrate.     

Mechanism of "L"-type pyruvate kinase from rabbit liver. Evidence against phosphoenzyme formation.

The "L"-type pyruvate kinase from rabbit liver does not catalyse exchange between phosphoenol[1-14C]pyruvate and pyruvate at either pH 8.5 or 6.2. Spectrophotometric experiments at pH 8.5 and 6;2 and gel-filtration experiments with [32P]phosphoenolpyruvate at pH 8,5 also fail to demonstrate phosphoenzyme formation. It is concluded that it is very unlikely that the enzyme has a phosphoenzyme mechanism.     

Correction to: Transmission of a novel predatory behaviour is not restricted to kin

Biological Invasions - A correction to this paper has been published: https://doi.org/10.1007/s10530-021-02548-x