Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1

SFTI-1 is a novel 14 amino acid peptide comprised of a circular backbone constrained by three proline residues, a hydrogen-bond network, and a single disulfide bond. It is the smallest and most potent known Bowman-Birk trypsin inhibitor and the only one with a cyclic peptidic backbone. The solution structure of [ABA(3,11)]SFTI-1, a disulfide-deficient analogue of SFTI-1, has been determined by H-1 NMR spectroscopy. The lowest energy structures of native SFTI-1 and [ABA(3,11)]SFTI-1 are similar and superimpose with a root-mean-square deviation over the backbone and heavy atoms of 0.26 +/- 0.09 and 1.10 +/- 0.22 Angstrom, respectively. The disulfide bridge in SFTI-1 was found to be a minor determinant for the overall structure, but its removal resulted in a slightly weakened hydrogen-bonding network. To further investigate the role of the disulfide bridge, NMR chemical shifts for the backbone H-alpha protons of two disulfide-deficient linear analogues of SFTI-1, [ABA(3,11)]SFTI-1[6,5] and [ABA(3,11)]SFTI-1[1,14] were measured. These correspond to analogues of the cleavage product of SFTI-1 and a putative biosynthetic precursor, respectively. In contrast with the cyclic peptide, it was found that the disulfide bridge is essential for maintaining the structure of these open-chain analogues. Overall, the hydrogen-bond network appears to be a crucial determinant of the structure of SFTI-1 analogues.

Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells

Prostate-specific antigen (PSA) and the related kallikrein family of serine proteases are current or emerging biomarkers for prostate cancer detection and progression. Kallikrein 4 (KLK4/hK4) is of particular interest, as KLK4 mRNA has been shown to be elevated in prostate cancer. In this study, we now show that the comparative expression of hK4 protein in prostate cancer tissues, compared with benign glands, is greater than that of PSA and kallikrein 2 (KLK2/hK2), suggesting that hK4 may play an important functional role in prostate cancer progression in addition to its biomarker potential. To examine the roles that hK4, as well as PSA and hK2, play in processes associated with progression, these kallikreins were separately transfected into the PC-3 prostate cancer cell line, and the consequence of their stable transfection was investigated. PC-3 cells expressing hK4 had a decreased growth rate, but no changes in cell proliferation were observed in the cells expressing PSA or hK2. hK4 and PSA, but not hK2, induced a 2.4-fold and 1.7-fold respective increase, in cellular migration, but not invasion, through Matrigel, a synthetic extracellular matrix. We hypothesised that this increase in motility displayed by the hK4 and PSA-expressing PC-3 cells may be related to the observed change in structure in these cells from a typical rounded epithelial-like cell to a spindle-shaped, more mesenchymal-like cell, with compromised adhesion to the culture surface. Thus, the expression of E-cadherin and vimentin, both associated with an epithelial-mesenchymal transition (EMT), was investigated. E-cadherin protein was lost and mRNA levels were significantly decreased in PC-3 cells expressing hK4 and PSA (10-fold and 7-fold respectively), suggesting transcriptional repression of E-cadherin, while the expression of vimentin was increased in these cells. The loss of E-cadherin and associated increase in vimentin are indicative of EMT and provides compelling evidence that hK4, in particular, and PSA have a functional role in the progression of prostate cancer through their promotion of tumour cell migration.

Mapping loss of heterozygosity in normal human breast cells from BRCA1/2 carriers

We have studied loss of heterozygosity at the BRCA1 and BRCA2 loci in 992 normal cell clones derived from topographically defined areas of normal tissue in four samples from BRCA1/BRCA2 mutation carriers. The frequency of loss of heterozygosity in the clones was low ( 1.01%), but it was found in all four samples, whether or not a tumour was present. Topographical mapping revealed that the genetic changes were clustered in some breast samples. Our study confirms the previous finding that a field of genetic instability can exist around a tumour, suggesting that sufficient tissue must be removed at surgery to avoid local recurrence. We also demonstrate that such a field of genetic change can exist in morphologically normal tissue before a tumour develops and, for the first time, we demonstrate that the field is of a size greater than one terminal duct-lobular unit. The genetic changes are not identical, however, which suggests that genetic instability in these regions may play an early role in tumour development. We also confirm and extend our original observation of loss of the wild-type BRCA1 allele in some clones, and loss of the mutant allele in others, demonstrating that loss of either allele is a stochastic event.

Selective loss of synaptic proteins in Alzheimer's disease: Evidence for an increased severity with APOE epsilon 4

A pathological feature of Alzheimer's disease (AD) is an area-specific neuronal loss that may be caused by excitotoxicity-related synaptic dysfunction. Relative expression levels of synaptopbysin, dynamin I, complexins I and II, N-cadherin, and alpha CaMKII were analysed in human brain tissue from AD cases and controls in hippocampus, and inferior temporal and occipital cortices. Synaptophysin and dynamin I are presynaptic terminal proteins not specific to any neurotransmitter system whereas complexin II, N-cadherin, and alpha CaMKII are specific for excitatory synapses. Complexin I is a presynaptic protein localised to inhibitory synapses. There were no significant differences in synaptophysin, dynamin I, N-cadherin, or alpha CaMKII protein levels between AD cases and controls. The complexin proteins were both markedly lower in AD cases than in controls (P < 0.01). Cases were also categorised by APOE genotype. Averaged across areas there was a 36% lowering of presynaptic proteins in AD cases carrying at least one epsilon 4 allele compared with in AD cases lacking the epsilon 4 allele. We infer that synaptic protein level is not indicative of neuronal loss, but the synaptic dysfunction may result from the marked relative loss of the complexins in AD, and lower levels of presynaptic proteins in AD cases with the APOE epsilon 4 allele. (c) 2006 Elsevier Ltd. All rights reserved.

The changing nature and role of the head of department

There is little doubt that devolution of responsibility to schools and the growth of school-based management have impacted upon the role and workload of school leaders. Not only Principals have been affected by these changes as Welch (1996) argues that Principals of public secondary schools have passed responsibility down to Deputy-principals and to Heads of Department. As such, the Head of Department role, like other school administration positions, has undergone significant change. Of interest to this paper is the changing role of Heads of Department in secondary schools. This study reports on the findings of semi-structured interviews with eight Heads of Department from four public secondary schools and Principals from each of these schools in South East Queensland. Four years after the first set of interviews, two heads of department were reinterviewed. Both sets of interviews focused upon the role, change, and the importance of leadership. The research generated eight specific themes each of which was considered consistent with the nature of the role in a period of significant cultural change. These were the difference in perceptions regarding the Head of Department role, held by Principals and Heads of Department; Head of Department leadership in terms of a curriculum framed department or whole school leadership; how individuals perceived leadership, and how they learned of leadership; the impact of the changing culture upon the individual Head of Department; the growing influence of situational factors upon the role; the impact of managerialism; the changing nature of a secondary school department; and a growing and more complex workload, and the need for different skills. Furthermore, the findings pointed towards the need for effective change processes and a reconceptualized head of department role. The paper concludes with some implications for the ongoing professional development needs of Heads of Department.

Progressive loss of expressive language in a journalist: What news for dementia?

A 77-year-old man with 8 year progressive language deterioration in the face of grossly intact memory was followed. No acute or chronic physiological or psychological event was associated with symptom onset. CT revealed small left basal ganglia infarct. Mild atrophy, no lacunar infarcts, mild diffuse periventricular changes registered on MRI. Gait normal but slow. Speech hesitant and sparse. Affect euthymic; neurobehavioral disturbance absent. MMSE 26/30; clock incorrect, concrete. Neuropsychological testing revealed simple attention intact; complex attention, processing speed impaired. Visuospatial copying and delayed recall of copy average with some perseveration. Apraxia absent. Recall mildly impaired. Mild deficits in planning, organization apparent. Patient severely aphasic, dysarthric without paraphasias. Repetition of automatic speech, recitation moderately impaired; prosody intact. Understanding of written language, nonverbal communication abilities, intact. Frontal release signs developed over last 12 months. Repeated cognitive testing revealed mild deterioration across all domains with significant further decrease in expressive, receptive language. Neurobehavioral changes remain absent to date; he remains interested, engaged and independent in basic ADLs. Speech completely deteriorated; gait and movements appreciably slowed. Although signs of frontal/executive dysfunction present, lack of behavioral abnormalities, psychiatric disturbance, personality change argue against focal or progressive frontal impairment or dementia. Relative intactness of memory and comprehension argue against Alzheimer’s disease. Lack of findings on neuroimaging argue against CVA or tumor. It is possible that the small basal ganglia infarct has resulted in a mild lateral prefrontal syndrome. However, the absence of depression as well as the relatively circumscribed language problem suggests otherwise. The progressive, severe nature of language impairments, with relatively minor impairments in attention and memory, argues for a possible diagnosis of primary progressive aphasia.

Management of a previously eroded tropical Alfisol with herbaceous legumes: Soil loss and physical properties under mound tillage

A study was carried out on a previously eroded Oxic Paleustalf in Ibadan, southwestern Nigeria to determine the extent of soil degradation under mound tillage with some herbaceous legumes and residue management methods. A series of factorial experiments was carried out on 12 existing runoff plots. The study commenced in 1996 after a 5-year natural fallow. Mound tillage was introduced in 1997 till 1999. The legumes - Vigna unguiculata (cowpea), Mucuna pruriens and Pueraria phaseoloides - were intercropped with maize in 1996 and 1998 while yam was planted alone in 1997 and 1999. This paper covers 1997-1999. At the end of each year, residues were either burned or mulched on respective plots. Soil loss, runoff, variations in mound height, bulk density, soil water retention and sorptivity were measured. Cumulative runoff was similar among interactions of legume and residue management in 1997 (57-151 mm) and 1999 (206-397 mm). However, in 1998, cumulative runoff of 95 mm observed for Mucuna-burned residue was significantly greater than the 46 mm observed for cowpea-burned residue and the 39-51 mm observed for mulched residues of cowpea, Mucuna and Pueraria. Cumulative soil loss of 7.6 Mg ha(-1) observed for Mucuna-burned residue in 1997 was significantly greater than those for Pueraria-mulched (0.9 Mg ha(-1)) and Mucuna-mulched (1.4 Mg ha(-1)) residues whereas in 1999 it was similar to soil loss from cowpea treatments and Pueraria-burned residue (2.3-5.3 Mg ha(-1)). There were no significant differences in soil loss in 1998 (1-3.2 Mg ha(-1)) whereas Mucuna-burned residue had a greater soil loss (28.6 Mg ha(-1)) than mulched cowpea (6.9 Mg ha(-1)) and Pueraria (5.4 Ms ha(-1)). Mound heights (23 cm average) decreased non-linearly with cumulative rainfall. A cumulative rainfall of 500 mm removed 0.3-2.3 cm of soil from mounds in 1997, 3.5-6.9 cm in 1998 and 2.3-4.6 cm in 1999, indicating that (detached but less transported) soil from mounds was far higher than observed soil loss in each year. Soil water retention was improved at potentials ranging from -1 to -1500 kPa by Mucuna-mulched residue compared to the various burned-residue treatments. Also, mound sorptivity at -1 cm water head (14.3 cm h(-1/2)) was higher than furrow sorptivity (8.5 cm h(-1/2)), indicating differences in hydraulic characteristics between mound and furrow. Pueraria-mulched residues for mounds had the highest sorptivity of 17.24 cm h(-1/2), whereas the least value of 6.96 cm h(-1/2) was observed in furrow of Mucuna-burned residue. Pueraria phas eoloides was considered the best option for soil conservation on the previously eroded soil, cultivated with mound tillage. (c) 2005 Elsevier B.V. All rights reserved.

Acyclic permutants of naturally occurring cyclic proteins - Characterization of cystine knot and beta-sheet formation in the macrocyclic polypeptide kalata B1

Kalata B1 is a prototypic member of the unique cyclotide family of macrocyclic polypeptides in which the major structural features are a circular peptide backbone, a triple stranded beta-sheet, and a cystine knot arrangement of three disulfide bonds. The cyclotides are the only naturally occurring family of circular proteins and have prompted us to explore the concept of acyclic permutation, i.e. opening the backbone of a cross-linked circular protein in topologically permuted ways. We have synthesized the complete suite of acyclic permutants of kalata B1 and examined the effect of acyclic permutation on structure and activity. Only two of six topologically distinct backbone loops are critical for folding into the native conformation, and these involve disruption of the embedded ring in the cystine knot. Surprisingly, it is possible to disrupt regions of the p-sheet and still allow folding into native-like structure, provided the cystine knot is intact. Kalata B1 has mild hemolytic activity, but despite the overall structure of the native peptide being retained in all but two cases, none of the acyclic permutants displayed hemolytic activity. This loss of activity is not localized to one particular region and suggests that cyclization is critical for hemolytic activity.

Presentation and mechanisms of concussion in professional Rugby League football

The present study prospectively recorded the circumstances, incidence, mechanisms, injury detection and presentation of concussion in Rugby League. Forty-three consecutive concussions were recorded over three competitive seasons in 175 professional Rugby League players. Data showed (i) the incidence of concussion ranged from 5.9 to 9.8 injuries/1000 player hours across grades - except when age-group players were mismatched (18.4); (ii) 'head-high tackles' accounted for a significant number of concussions; (iii) concussion rarely involved a loss of consciousness with the most common indicators of concussion being amnesia, headache and unsteadiness, with the mechanism of injury often missed; and (iv) concussion often occurs concurrently with other injuries. Concussion (including repeated episodes) is a common injury in Rugby League. Systematic mental status questioning is warranted whenever concussion is suspected. Coaches, trainers and players need more education in the recognition and management of concussion. Stricter penalties for illegal 'head-high' tackling are strongly recommended.