Alpha-1-antitrypsin and transferrin null alleles in the Portuguese population

In a Portuguese family, a null allele was found in the Pi system. An apparent 'exclusion' of the mother was found to be due to the presence of null alleles in mother and child. A transferrin (Tf) null allele was found in a case of disputed paternity. The mother and putative father were heterozygous for Tf null alleles and the child was homozygous (TfQ0) and presented hypotransferrinemia.     

Transferrin subtypes and variants in Germany; Further evidence for a Tf null allele

Summary Isoelectric focusing (IEF) with carrier ampholytes was used for the determination of transferrin C subtypes and transferrin B and D variants in a sample of 1125 unrelated individuals from Southern Germany. The observed TfC allele frequencies were Tf*C1=0.7872, Tf*C2=0.1365, and Tf*C3=0.0675. The rare C subtype C6 was observed twice. A new C subtype, called C10, was observed and identified by IEF with immobilized pH gradients. The rare C subtypes C4 and C8 were also studied by this method. TfB and TfD variants were found with a heterozygous frequency of 1.53%. One new TfD was found which is located between D1 and D2 and therefore named D1-2. Evidence for a Tf null allele was obtained in a child and the putative father; they were considered to be heterozygous for an allele Tf0. The theoretical exclusion rate for paternity examinations was calculated for the Tf system and found to be 17.95%.     

The costs of human locomotion: Maternal investment in child transport

This article investigates maternal investment in child carrying and presents a method for determining when it is energetically advantageous for a mother to carry her child rather than force her child to walk independently. I calculate maternal and child energy consumption while walking and develop correction factors to facilitate making these energy calculations for young children. In addition, I investigate the effect of maternal burdens in addition to the child and of external nutritional support on energy consumption. Since maternal energy is a finite resource, the “decision” to carry a child or force it to walk independently is especially important. This decision can be predicted from the body mass of the mother and child and the child's age. If the mother provides all of the child's nutrition, then the mother should choose to carry her child only when the energy usage of the mother carrying the child is less than the sum of the energy used when the mother and child walk independently. The critical velocity, when the two expenditures are equal, can then be determined. Several general hypotheses are also addressed. The critical velocity of a 60 kg mother with a 4-year-old child approximately equals the average walking speed of adult humans. For a lighter mother, the critical velocity is reached when her child is 3 years old, while for heavier mother this point is not reached until her child is 6 years old. The effect of burdens in addition to the child's mass is minimal. Nutritional support of the child by agencies other than the mother decreases the age at which the mother should force the child to walk independently. In some cases, especially for the lightest mothers, it is never in the mother's best energetic interest to carry her child. Am J Phys Anthropol 107:71–85, 1998. © 1998 Wiley-Liss, Inc.     

Formal genetics of esterase D (EC 3.1.1.1): evidence for a sex-phenotype association

The formal genetics of esterase D (EC 3.1.1.1) was studied in family data and mother/child pairs. A general agreement with mendelian expectations was found. However, a significant sex-phenotype association was detected in families from northwestern Portugal as well as in mother/child pairs and family data from southwestern Germany.     

Oligomerized transferrin receptors are selectively retained by a lumenal sorting signal in a long-lived endocytic recycling compartment

Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (Tf10) which was prepared by chemical cross- linking of transferrin (Tf). Tf10 was internalized about two times slower than Tf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of Tf10 was investigated using fluorescence and electron microscopy. Tf10 was not delivered to the lysosomal pathway followed by low density lipoprotein but remained accessible to Tf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained Tf10 was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tf10. The presence of Tf10 within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tf10 within the recycling compartment did not require the cytoplasmic domain of the TfR since Tf10 exited cells with the same rate when bound to the wild-type TfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by Tf10 is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf.     

Transferrin overexpression alters testicular function in aged mice

Many studies have shown a correlation between transferrin (Tf) concentration and sperm yield in several mammalian species. We have used transgenic mice expressing human Tf (hTf) to investigate if overexpression of Tf increases the efficiency of mouse spermatogenesis. We demonstrated that a 36% increase of Tf does not ameliorate the efficiency of mouse spermatogenesis but on the contrary resulted in a 36% decrease of testis sperm reserves. Tf overexpression had no effect on testicular determination and development, however testicular function of these transgenic mice was affected in an age-dependent manner. At 16 months of age, testicular and epididymal weights were significantly reduced. While spermatogenesis was qualitatively normal, testicular functions were perturbed. In fact, testosterone rate after human chorionic gonadotropin (hCG) stimulation was lower in Tf overexpressing mice. Intratesticular concentration of estradiol-17beta was increased and fluid accumulation after ligation of rete testis was more abundant in these transgenic mice. Surprisingly, we found that endogenous Tf levels were also increased in Tf overexpressing mice and we demonstrated for the first time that Tf may serve to upregulate its own expression in testis. Collectively, our data show that Tf overexpression has negative effects on testicular function and that Tf levels require strict regulation in the testis.     

A new transferrin variant from Iran (Tf B-Iran): review of 36 variant alleles

A total of 2581 serum samples collected from five population groups of Iran was studied for electrophoretic variations of the transferrin (Tf). Besides the common phenotype Tf C the authors could observe 41 individuals with rare Tf types: CB1, CB2, CD1, CDChi, CD2. In addition to these Tf types two individuals with a new Tf B variant were observed. This new variant was found in the Dezfooli sample and was designated as Tf B-Iran. The electrophoretic position of this variant is described, and all the hitherto known Tf variants are reviewed.     

Uptake of diterbium transferrin, a potential multi-photon-excited microscopy probe, into human leukemia K562 cells via a transferrin-receptor-mediated process

Our present study provided new evidence for diterbium transferrin (Tb₂Tf) as a potential multi-photon-excited microscopy probe. It indicated that the Tb₂Tf complex can be transported into human leukemia K562 cells via a process mediated by transferrin (Tf) receptors as an intact entity and with no obvious cellular toxicity. The supporting evidence includes the following. First, the transport kinetic behavior of Tb was compared with that of the Tf moiety. The Tb was determined by inductively coupled plasma mass spectrometry and Tf was determined by fluorescence activated cell sorting analysis. The kinetic synchronization of internalization of both Tb and Tf into human leukemia K562 cells demonstrated the Tb₂Tf complex was transported into cells as a whole. Second, using confocal laser scanning microscopy, we observed the localization of Tb₂Tf in the cell. This showed that the internalized Tb₂Tf was mostly situated in the same perinuclear region as diferric transferrin (Fe₂Tf). In addition, pretreatment with pronase largely abolished the transport process of Tb₂Tf. The relative fluorescence intensities representing the uptake of Tf into the cells decreased to about 16% and the cytosolic Tb content decreased to almost the same percentage as for Tf. Furthermore, the addition of Fe₂Tf can effectively inhibit transport of Tb₂Tf into K562 cells. Third, no significant decrease of cell viability was observed in the presence of Tb₂Tf even for 24 or 48 h by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Moreover, prospects for the use of Tb₂Tf as a multi-photon-excited microscopy probe in a living system are discussed.     

Comparative studies of the binding and growth-supportive ability of mammalian transferrins in human cells

The ability of human-derived cells in culture to bind, remove iron from, and grow in the presence of transferrins (Tf) isolated from the sera of species commonly included in tissue culture medium was investigated. Kinetic studies on HeLa cells reveal apparent first-order association rate constants of 0.43 min-1 for human Tf and 0.15 min-1 for equine Tf. Labeled chicken ovo-Tf and fetal bovine Tf were not recognized by the HeLa cells. Competition experiments with HeLa cells that use either isolated Tf or parent serum confirm these findings. Equilibrium binding experiments performed on HeLa cells at 37 degrees C in the presence of 2,4-dinitrophenol to prevent iron removal indicate 1 X 10(6) Tf bound/cell with a dissociation constant (K'D) of 28 nM for human Tf and 182 nM for equine Tf. Equilibrium binding performed at 0 degrees C to prevent endocytosis reveals 4.1-6.7 X 10(5) Tf binding sites/cell with a K'D of 8.3 nM for human Tf and 41.5 nM for equine Tf. Parallel experiments in normal human diploid fibroblast-like MRC-5 cells indicate expression of 0.82-2.78 X 10(5) Tf binding sites/cell with a K'D of 8.2 nM for human and 39.1 nM for equine Tf. Thus, the results of equilibrium binding studies of a more differentiated cell type are consistent with those found for HeLa cells. Fetal bovine Tf was found to compete weakly with labeled human Tf for human receptor on HeLa cells in a soluble receptor assay, with an approximately 500-fold excess needed to reduce binding to half maximal. Iron uptake experiments show an iron donating hierarchy where human greater than horse greater than calf, suggesting that the rate of iron uptake depends on the affinity of receptor for transferrin. Growth experiments involving HeLa cells in chemically defined serum-free medium demonstrate that bovine Tf will support growth as well as human Tf, but at concentrations much higher than are required of human Tf.     

Transferrin in cultured human term cytotrophoblast cells: Synthesis and heterogeneity

Transferrin (Tf) mRNA was recently demonstrated in rat and mouseplacental tissue. Rat placental cells were shown to secrete transferrin. Thecell type with which Tf mRNA was associated was not investigated. Wetherefore studied the ability of immunopurified human term cytotrophoblastcells in culture to synthesize Tf, by means of pulse-label experiments with35S-methionine and report that these cells do synthesize Tf. Tf mRNA wasdemonstrated in the cell lysates by means of RT-PCR. Tf isolated fromcytotrophoblast and syncytiotrophoblast cells was shown to be different fromboth maternal and fetal serum Tf with respect to the distribution ofisoforms as demonstrated by means of iso-electric focusing. The iso-electricpoints were found at lower pH values (pH 5.0-5.4), compared to theiso-electric points of maternal and fetal serum Tf, suggesting a higherdegree of sialylation and glycan chain complexity.     

Binding to cellular receptors results in increased iron release from transferrin at mildly acidic pH

In order to better understand the cellular delivery of iron from serum transferrin (Tf), we compared iron release from receptor-bound and free Tf. While free Tf did not release all iron until below pH 4.6, receptor-bound Tf released significantly more iron at mildly acidic pH, with essentially all iron released between pH 5.6 and 6.0. Since Tf is acidified to a minimum pH of 5.4 in K562 cells, this result accounts for the nearly complete extraction of iron from Tf by these cells. Comparison of fluorescence from Tf conjugated with lissamine rhodamine sulfonyl chloride (LRSC-Tf) free in solution and bound to receptor provides further evidence that the Tf receptor modulates low pH-mediated conformational changes in Tf. As pH was decreased from neutrality, the fluorescence of free LRSC-Tf began to increase below pH 6.2; the fluorescence of LRSC-Tf bound to human receptors did not increase until below pH 5.6. Binding to the Tf receptor, while facilitating iron release from Tf, appears to partially inhibit a conformational change that causes the increase in LRSC-Tf fluorescence at low pH. The fluorescence of human LRSC-Tf bound to murine receptors increases at a higher pH, 6.0, indicating that there are differences in conformational stabilization of Tf by receptors of different species. The results suggest that the Tf receptor, in addition to providing a means by which cells may internalize Tf, functions to increase the release of iron from Tf in the endosome.     

Evidence for the Tf locus being associated with an early lethal factor in a strain of pigs

The frequencies of three co-dominant alleles Tf ^A, Tf ^B and Tf ^c controlling the serum transferrin locus in native and exotic breeds of pig in the United Kingdom are shown. Matings between different transferrin genotypes and segregation of the transferrin alleles in piglets from 131 litters are also recorded.
In eleven litters from matings within a closely related group of animals heterozygous for the Tf ^c allele, no offspring of the Tf ^c/ Tf ^c genotype were born. Matings between heterozygous related boars believed to be carrying a lethal factor linked to the Tf locus and heterozygous unrelated sows of the Saddleback breed, resulted in offspring of the Tf ^c/ Tf ^c genotype being born.
An excess of heterozygote genotypes was found in all litters from matings between animals heterozygous for the Tf ^c allele. In all these litters the average litter size was 0.5 piglets less per litter than the average for all other litters studied. For the eleven litters from matings involving closely related animals heterozygous for the Tf ^c allele, the average litter size was 1.4 piglets less per litter than the average for all other litters.
The possibility of an early lethal factor being linked to the Tf locus in this family of pigs is discussed.     

The monotony of transferrin and esterase electrophoretic patterns in pirarucu, Arapaima gigas (Schinz, 1822) from Santa Cruz Lake, Tefé River, Amazonas, Brazil

Starch gel electrophoresis was used for examining the transferrin gene locus (Tf) and two esterase gene loci (Est-1 and Est-D1) of a pirarucu (Arapaima gigas) population sample collected from Santa Cruz Lake, Tefé River, Amazonas, Brazil. The Tf locus was tentatively classified as being polymorphic, showing two double-banded patterns (Tf(12) and Tf(22)) of the three theoretically expected ones (Tf(11), Tf(12) and Tf(22)), presumably controlled by two co-dominant alleles, Tf(1) and Tf(2). The monotony detected in pirarucu Tf locus genotypes showing a very high proportion of the double-banded heterozygote pattern Tf(12) (95% of the sampled individuals) may indicate the possibility of their having come from representatives of the same brood begotten by a pair of fish, where a single-banded Tf(11) homozygote pattern male would have crossed with a single-banded Tf(22) homozygote pattern female, or vice versa. One zone of electrophoretic activity was detected in esterase, presumably controlled by a monomorphic Est-1 locus with the fixed allele Est-1(1) where all individuals showed the single-banded Est-1(11) homozygote pattern. Esterase-D also displayed one zone of electrophoretic activity, presumably controlled by a monomorphic Est-D1 locus with a fixed allele Est-D1(1) where all individuals revealed the single-banded Est-D1(11) genotype pattern. The monotony comprised by single-banded genotype patterns in both esterase systems tested may also indicate the possibility of the individuals from the sample examined having come from representatives of the same brood begotten by a pair of fish with both the male and female having the same genotypes.     

Relationship between rabbit transferrin electrophoretic patterns and plasma iron concentrations

Rabbit transferrin (Tf) was studied electrophoretically using 1141 blood samples from individuals belonging to seven populations (Spanish Common, Spanish Giant, Butterfly, Lyoné de Bourgogne, New Zealand White, Californian and New Zealand White X Californian hybrids). No Tf polymorphism was found by starch gel electrophoresis, but six patterns, differing in the presence and/or intensity of three bands ('a', anodic; 'b', intermediate; and 'c', cathodic) were observed by polyacrylamide gel electrophoresis. No genetic model could explain these patterns, since they reflect differences in plasma Tf iron content. The electrophoretic test allowed a direct observation of the relative in vivo levels of the different Tf molecular species; saturated (band 'a', Fe2Tf); semi-saturated (band 'b', Fe1Tf); and without iron (band 'c' Fe0Tf, apotransferrin). The degree of iron saturation of Tf varied among individuals and throughout the individual's life. Specifically, in pregnant females, Fe2Tf and Fe1Tf are generally observed, except in late pregnancy (from day 25 to parturition), when mainly apotransferrin is observed. Significantly, within 24 h post-partum, high levels of Fe2Tf are reached in the female's serum.     

The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum

We used a conjugate of transferrin and horseradish peroxidase (Tf/HRP) to label the intracellular transferrin receptor route in the human hepatoma cell line HepG2. The recycling kinetics of [125I]Tf/HRP were similar to those of unmodified [125I]Tf, implying identical routes for both ligands. 3,3'Diaminobenzidine (DAB)-cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were incubated with both Tf/HRP and [125I]Tf, and caused two different effects: (a) the equilibrium density of [125I]Tf containing microsomes in a Percoll density gradient was increased, and (b) the amount of immunoprecipitable [125I]Tf from density-shifted lysed microsomes was only 20% of that of nonDAB treated microsomes. The whole biosynthetic route of alpha 1-antitrypsin (AT), a typical secretory glycoprotein in HepG2 cells, was labeled during a 60-min incubation with [35S]methionine. DAB cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were also incubated with Tf/HRP. DAB cytochemistry caused approximately 40% of microsome- associated "complex" glycosylated [35S]alpha 1-antitrypsin ([35S]c-AT) to shift in a Percoll density gradient. Only part of the density shifted [35S]c-AT could be recovered by immunoprecipitation. A maximum effect was measured already after 10 min of Tf/HRP uptake. The density distribution of the "high mannose" glycosylated form of 35S-alpha 1- anti-trypsin [( 35S]hm-AT) was not affected by Tf/HRP. If in addition to Tf/HRP also an excess of non-conjugated transferrin was present in the medium, [35S]c-AT was not accessible for Tf/HRP, showing the involvement of the transferrin receptor (TfR) in the process. Furthermore, we show that if Tf/HRP and [35S]c-AT were located in different vesicles, the density distribution of [35S]c-AT was not affected by DAB-cytochemistry. Pulse-labeling with [35S]methionine was used to show that [35S]c-AT became accessible to endocytosed Tf/HRP minutes after acquirement of the complex configuration. A common intracellular localization of endocytosed Tf/HRP and secretory protein could be confirmed by immuno-electron microscopy: cryosections labeled with anti-albumin (protein A-colloidal gold) as well as DAB reaction product showed double-labeling in the trans-Golgi reticulum.     

Two new sheep transferrin variants and the effect of neuraminidase

Transferrin phenotypes were determined in six breeds of sheep by starch gel electrophoresis. Two new variants, Tf H_Czech and Tf K_Czech, were found and some evidence of their genetic control was obtained. Tf H_Czeeh was detected only in Sumava sheep; it has an intermediate mobility between Tf A and Tf B. Tf K_Czech was found only in Tsigais; it was localized between Tf B and Tf C. The frequencies of corresponding alleles were very low.
Individual transferrin variants (I, A, H_Czech, B, K_czech, C, D, E, and P) were treated with neuraminidase. Electrophoretic mobility of the strong band was decreased by two steps in each case. It suggests that in the strong Tf band two sialic acid residues are accessible to the enzyme.     

Absence of Binding Between the Human Transferrin Receptor and the Transferrin Complex of Biological Toxic Trace Element,Aluminum, Because of an Incomplete Open/Closed Form of the Complex

Human transferrin (Tf) very tightly binds two ferric ions to deliver iron to cells. Fe(III)₂Tf (Fe₂Tf) binds to the Tf receptor (TfR) at pH 7.4; however, iron-free Tf (apoTf) does not. Iron uptake is facilitated by endocytosis of the Fe₂Tf–TfR complex. Tf can also bind aluminum ions, which cause toxic effects and are associated with many diseases. Since Al(III)₂Tf (Al₂Tf) does not bind to TfR, the uptake of aluminum by the cells does not occur through a TfR-mediated pathway. We have studied the absence of binding between Al₂Tf and TfR by investigating the physicochemical characteristics of apoTf, Al₂Tf, Fe₂Tf, and TfR. The hydrodynamic radius of 38.8 Å for Al₂Tf obtained by dynamic light scattering was between that of 42.6 Å for apoTf and 37.2 Å for Fe₂Tf. The ζ potential of ?11.3 mV for Al₂Tf measured by capillary electrophoresis was close to ?11.2 mV for apoTf as compared to ?11.9 mV for Fe₂Tf, indicating that the Al₂Tf surface had a relatively scarce negative charge as the apoTf surface had. These results demonstrated that the structure of Al₂Tf was a trade-off between the closed and open forms of Fe₂Tf and apoTf, respectively. Consequently, it is suggested that Al₂Tf cannot form specific ionic interresidual interactions, such as those formed by Fe₂Tf, to bind to TfR, resulting in impossible complex formation between Al₂Tf and TfR.     

Evidence for nonvectorial, retrograde transferrin trafficking in the early endosomes of HEp2 cells

We have previously characterized the trafficking of transferrin (Tf) through HEp2 human carcinoma cells (Ghosh, R. N., D. L. Gelman, and F. R. Maxfield, 1994. J. Cell Sci. 107:2177-2189). Early endosomes in these cells are comprised of both sorting endosomes and recycling compartments, which are distinct separate compartments. Endocytosed Tf initially appears in punctate sorting endosomes that also contain recently endocytosed LDL. After short loading pulses, Tf rapidly sorts from LDL with first-order kinetics (t1/2 approximately 2.5 min), and it enters the recycling compartment before leaving the cell (t1/2 approximately 7 min). Here, we report a second, slower rate for Tf to leave sorting endosomes after HEp2 cells were labeled to steady state with fluorescein Tf instead of the brief pulse used previously. We determined this rate using digital image analysis to measure the Tf content of sorting endosomes that also contained LDL. With an 11-min chase, the Tf in sorting endosomes was 24% of steady-state value. This was in excess of the amount expected (5% of steady state) from the rate of Tf exit after short filling pulses. The excess could not be accounted for by reinternalization of recycled cell surface Tf, implying that either some Tf was retained in sorting endosomes, or that Tf was delivered back to the sorting endosomes from the recycling compartment. The former is unlikely since nearly all sorting endosomes contain detectable Tf after an 11-min chase, even though more than one third of the sorting endosomes were formed during the chase time. Furthermore, while observing living cells by confocal microscopy, we saw vesicle movements that appeared to be fluorescent Tf returning from recycling compartments to sorting endosomes. The slow rate of exit after steady-state labeling was similar to the Tf exit rate from the cell, suggesting an equilibration of Tf throughout the early endosomal system by this retrograde pathway. This retrograde traffic may be important for delivering molecules from the recycling compartment, which is a long-lived organelle, to sorting endosomes, which are transient.