A comparative study in methods of cryoprotection for human semen

A comparative study using either glycerol or an egg yolk-citrate-glycerol mix for cryoprotection under the conditions described showed the latter to give a significantly better post-thaw motility. The greatest drop was noted within the first hour, suggesting that freezability of a sample could be judged accurately by rethawing at that time.Controlled studies using scanning electron microscopy clearly showed a greater head disruption after freezing with glycerol and looped tails after centrifugation, which could account for some of the findings of the first part of the study.These findings are reviewed and it is recommended that seminal samples are not centrifuged and that a complex medium be employed for seminal freezing and storing.     

Internal intensity standards for heme protein UV resonance Raman studies: excitation profiles of cacodylic acid and sodium selenate.

We examine the utility of SO4(2-), ClO4-, cacodylic acid, and SeO4(2-) as internal intensity standards for Raman spectral measurements of protein structure. We find that 0.1 M SO4(2-) and ClO4- perturb the protein tertiary structure of aquomethemoglobin (met-Hb) and its fluoride (met-HbF) and azide (met-HbN3) complexes. Changes occur for the tryptophan near-UV absorption bands, the iron spin state is altered, and the fluoride ligand affinity decreases. Concentrations of ClO4- and SO4(2-) as low as 0.1 M suppress the met-HbF quaternary R----T transition induced by the allosteric effector inositol hexaphosphate (IHP). In contrast, similar concentrations of cacodylic acid and SeO4(2-) show little effect on the hemoglobin tertiary or quaternary protein structures or upon the R----T transition induced by IHP. We measure the Raman cross sections of cacodylic acid and SeO4(2-) between 218 and 514.5 nm and find that for UV excitation they are ca. 5-fold larger than ClO4- or SO4(2-). Thus, cacodylic acid and selenate can be used at lower concentrations. Cacodylic acid and SeO4(2-) are superior Raman internal intensity standards for protein structural studies.     

Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin-like growth factor (IGF)-1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF-1/insulin induced production of keratinocyte-derived autocrine growth factors that bind to the EGF receptor. Increased steady-state mRNA levels for transforming growth factor alpha (TGF-α) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF-1. IGF-1 also induced production and secretion of TGF-α and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF-1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth by IGF-1 requires activation of an EGF receptor-mediated autocrine loop. © 1995 Wiley-Liss, Inc.     

Complete amino acid sequence of a novel integrin beta subunit (beta 6) identified in epithelial cells using the polymerase chain reaction

The integrin family of adhesion receptors consists of several heterodimeric glycoproteins, each composed of one alpha and one beta subunit. Three different mammalian beta subunits, beta 1, beta 2, and beta 3, have been sequenced, but recent evidence suggests the existence of several others. Amplification of guinea pig airway epithelial cell cDNA with oligonucleotide primers designed to recognize consensus integrin beta subunit sequences led to the identification of a novel partial cDNA sequence. Clones containing portions of this sequence were used to screen cDNA libraries constructed from the human pancreatic carcinoma cell line FG-2 and identified a series of overlapping clones encoding the full-length sequence of the human homologue of this protein. This sequence of 788 amino acids is 43, 38, and 47% identical to the sequences of beta 1, beta 2, and beta 3, respectively. Features shared between this novel protein and the previously sequenced beta subunits include the positions of all 56 cysteine residues in the extracellular domain, the single putative transmembrane domain, and the short putative cytoplasmic domain. However, a unique 11-amino acid extension at the carboxyl terminus, not present in any of the other beta subunits, is suggestive of distinctive interactions with cytoplasmic components. Comparison of the human and guinea pig sequences reveals a high degree (94%) of cross-species conservation. Because this protein is clearly distinct from the two other recently described integrins beta 4 and beta 5, we propose to designate it beta 6.     

The effects of soluble-Al on root growth and radicle elongation

Summary In order to determine the effects of concentration on plant growth, aluminium (Al) was extracted (10^–3 M CaCl₂) from 4 acid brown hill soils which had been treated with superphosphate at rates equivalent to 0 to 300 kg P ha^–1. The soils ranged in pH (CaCl₂) from 3.5 to 4.9, and Al concentration from 0 to 0.6 mM. The effects of Al on ryegrass growth in the 4 soils in a glasshouse was compared with its effect on radicle elongation of seeds germinated in contact with CaCl₂ extracts from the same soils.Ryegrass root growth in the glasshouse, and radicle elongation in the bioassay test were both unaffected by Al concentrations below 0.1 mM. Root growth was substantially reduced when Al concentration exceeded 0.1 mM and above 0.2 mM growth was almost completely inhibited. Radicle elongation rate was also reduced when the concentration of Al was greater than 0.2 mM agreeing well with the observation from the pot experiment.It is concluded that because of its speed and convenience the bioassay method offers a useful method of establishing critical levels of Al for crop plants.