Male fertility has been decreasing in recent decades. In rats, a diet rich in plant oestrogens (phytoestrogens) from birth results in adult testes that have reduced germ cells (spermatogonia) and germ supporting cells (Sertoli cells). This project aimed to determine when in development this reduction in Sertoli cells and spermatogonia occurs.

Male rats and their mothers were fed a high phytoestrogen (456µg/g isoflavones) or phytoestrogen-free diet from conception to death. Groups of phytoestrogen-free and high phytoestrogen treated male rats were killed at 5 (n = 10) and 18 (n = 10) days post-partum. Their testes were removed, embedded in wax, cut into 30µm sections, and stained with haematoxylin and eosin. The Sertoli cells, Leydig cells, and spermatogonia were counted using the optical disector method, combined with the Cavalieri method. Another group of phytoestrogen-free and high phytoestrogen treated rats (n = 10) were injected with bromodeoxyuridine (BrdU) two hours prior to death at 18 days post-partum. Their testes were removed, embedded in wax, cut into 5µm sections, and then stained using immunohistochemistry. Immuno-positive staining cells were counted using the physical disector method, combined with the Cavalieri method.

Rats fed a high phytoestrogen diet had fewer Sertoli cells than those fed a phytoestrogen-free diet at 18 (Student’s t-test, P = 0.0435), but not at five, days post-partum. There was no significant difference in Leydig cells or spermatogonia between groups at either age, and no significant difference in cell proliferation (BrdU immuno-positive staining cells) between groups at 18 days. These data show that Sertoli cells are reduced between 5 and 18 days post-partum, and any reduction in spermatogonia occurs after 18 days in rats. If similar changes occur in human males it could be important to reduce children’s exposure to environmental oestrogens, as this may improve fertility.

Tooth extraction may lead to the development of osteoradionecrosis (ORN) in irradiated jaws. Hyperbaric oxygen therapy (HBOT) is used to prevent ORN by applying steep oxygen gradients to the irradiated tissues making them less hypocellular, hypovascular and hypoxic. The main aim of the study was to review the outcome, through a retrospective clinical audit, of the use of prophylactic HBOT to prevent the development of ORN as a result of dental extractions in patients who had received head and neck irradiation between 2003 and 2006. An additional aim was to assess the incidence of reported visual changes following HBOT.

Information was collected from clinical records at the Greenlane Clinical Centre, Auckland. It comprised patients’ age, gender, site and type of tumour, amount of radiation dose received in Gray, time lapse between radiotherapy and dental extraction, date of 20th HBOT treatment and the HBOT protocol used in patients. Diagnosis of ORN, time taken to heal following extraction, pain intensity and incidence of visual changes was obtained through a telephone interview survey with patients using a standardized quality of life questionnaire.

Twenty-six out of 29 patients (90%) were interviewed, of whom 25 (96%) were successfully treated with only one (4%) developing ORN. Nine of the 26 (35%) HBOT patients developed visual changes which lasted for a maximum of eight weeks post-HBOT (as predicted).

It appears to be beneficial to use HBOT prophylactically to prevent the development of ORN while carrying out traumatic dental work, particularly extractions, following radiotherapy to the head and neck. Visual changes following the 20th HBOT treatment appear to be transient and, if present, last for a maximum of 8 weeks.

Species of the genus Streptococcus constitute the numerically dominant bacteria on the teeth of almost all humans. Streptococci are genotypically diverse and isolates cultured from bite marks may be matched to those from the teeth of the biter. These characteristics potentially provide evidence from bite mark investigations in which human DNA cannot be recovered. This study investigated the feasibility of applying PCR and denaturing gradient gel electrophoresis (DGGE) as a non-culturing approach to compare streptococcal DNA from bite marks and teeth.

Swabbed samples were taken from self-inflicted bite marks (three hours old), unbitten skin sites and the mandibular incisors of 24 participants. Extracted DNA was amplified by PCR using streptococcus-specific primers and the products separated by DGGE. PCR products from various laboratory strains provided reference markers. Relative migration distances of amplicons were measured manually. Following exclusion of skin amplicons, the number of DNA fragments amplified from bite marks that matched those from the teeth of the same and different individuals were determined.

The mean percentage match of amplicons from bite marks with those from the tooth samples (19 comparisons) of the same individual was 82.8% (95% confidence interval, 58.2% - 100.0%). The mean percentage match between bite marks and tooth samples (345 comparisons) of different individuals was 36.2% (95% confidence interval, 2.9% - 69.5%). A significant difference was found between the two groups (P < 0.0001; Student t-test).

The results indicate a high likelihood of matching bacterial DNA amplified directly from a bite mark with bacterial DNA from the teeth responsible for the bite mark. Although some bite marks displayed a number of amplicons matching heterologous teeth, refinement of the method, including additional PCR primers and computer-assisted analysis, should enhance the specificity of this approach.

Lipoprotein(a) [Lp(a)] is a low density lipoprotein-apolipoprotein(a) complex present in plasma that is associated with cardiovascular disease. Lp(a) concentration is largely determined by the apo(a) gene. Single nucleotide polymorphisms (SNPs) in this gene can lower Lp(a) concentration and cause null apo(a) phenotypes. A significant proportion of null apo(a) phenotypes in populations studied to date, however, remain genetically uncharacterised. We recently discovered a new SNP in two apo(a) null individuals located at the apo(a) KIV-10 exon 1 +1 donor splice site (KIV-10 ex1 +1 G→A) that was similar to another previously reported null apo(a) SNP. We investigated the frequency of the KIV-10 ex1 +1 G→A SNP in a local population previously shown to have a significant number of null apo(a) phenotypes to investigate any relationship between the two.

A restriction fragment length polymorphism (RFLP) assay for the KIV-10 ex1 +1 G→A SNP was designed and applied to 164 samples from the control group of the Otago Vascular Disease population. Primers were designed to amplify a 125 bp sequence surrounding the KIV-10 +1 donor splice site and create a DdeI restriction site so that presence of the KIV-10 ex1 +1 G→A SNP could be determined following PCR and restriction digestion.

Using this assay, 61% of samples were genotyped as wild type individuals and 37% of samples were genotyped as heterozygotes. No homozygotes were detected. The median Lp(a) concentration for heterozygous individuals was 14.94 nmolL-1 and for wild type individuals was 15.74 nmolL-1. A Mann-Whitney U Rank statistical test showed no significant difference (P = 0.6904) between these values suggesting no association between the KIV-10 ex1 +1 G→A SNP and Lp(a) levels and likely no association between this SNP and the null apo(a) phenotype.

In conclusion, although the KIV-10 exon 1 +1 donor splice site SNP is certainly frequent in this population, it does not appear to be associated with the null apo(a) phenotype.

Fluconazole (FLC) has become the antifungal drug of choice for non-life-threatening Candida infections. However, there is an increasing clinical problem of drug resistance. This frequently results from elevated expression of the major Candida albicans ATP-binding cassette (ABC) drug efflux pumps Cdr1p and Cdr2p in the yeast cell membrane. This study examined the function of two putative phosphorylation sites in the C. albicans drug efflux pump Cdr1p by undertaking site-directed mutagenesis and expression of mutant C. albicans Cdr1p in the heterologous host, Saccharomyces cerevisiae.

Site-directed mutagenesis was accomplished by recombinant PCR in which the amino acid serine (S), at the putative phosphorylation sites S312 and S487, was changed to either alanine (A) or aspartate (D). The template for the recombinant PCRs was a plasmid containing the CDR1B allele of C. albicans ATCC10261. Cassettes were constructed comprising the mutant CDR1 gene, a selection marker (URA3) and flanking S. cerevisiae gene sequences and were used to transform the host strain to ura+. The effects of the introduced mutations were determined using functional assays of FLC susceptibility, and rhodamine 6G (R6G) efflux.

The S487A(D) mutants had FLC susceptibilities and R6G efflux activities similar to the strain expressing the parental CDR1 allele, with a minimal inhibitory concentration (MIC) of 300 and 200 respectively. The S312A(D) mutants showed increased susceptibilities to FLC, and decreased R6G efflux.

The double S312A/S487A mutant had properties similar to S312A mutant with an MIC of 150. The double S312D/S487D mutant was the most affected with the least resistance to FLC with an MIC of 100, and reduced R6G efflux activity.

In summary, the mutation of S312A but not S487A affected Cdr1p functions and the S312D/S487D double mutation gave the greatest reduction in FLC resistance and R6G efflux, suggesting a mutation at both sites could help reduce the increasing problem of drug resistance.

The striatum is an area of the brain that has a role in movement and reward-related behaviour. Medium spiny neurons, the main output cells of the striatum, make up more than 95% of the total striatal cell population. However interneurons have an important regulatory role in the striatum. Cholinergic interneurons receive inputs from the cerebral cortex and thalamus at distal and proximal dendrites, respectively, and signal when a relevant behavioural event occurs. The basis of their ability to mediate this signalling lies in their intrinsic cell properties and is influenced by the morphology and electrical mechanisms of the dendrites. The aim of this project was to model some of the intrinsic properties of the cholinergic cell using the online computer simulation platform NEURON.

We used the 3-D morphology of a neuron with a similar morphology to a striatal cholinergic interneuron as the basis of a compartmental model. The cell chosen was a Cajal-Retzius neuron found in layer I of the cerebral cortex. NEURON was used to model 80 compartments including 66 dendritic sections. Individual currents were modelled using NMODL, NEURON’s model description language. Six main currents were modelled: passive current, potassium delayed rectifier current, A-Type potassium current, sodium current, cationic hyperpolarising current (Ih) and inward rectifying potassium current. Ion channel subunit composition was assessed by examining published reverse-transcriptase PCR and immunohistochemistry studies.

Using empirically determined individual currents obtained from the literature, we were able to successfully replicate electrophysiological tracings recorded directly from cholinergic neurons in the literature and from our own laboratory. We determined that the type of channel which best described the Ih current was the hyperpolarisation-activated cyclic nucleotide-gated potassium channel 4 (HCN4) subtype. This model will inform future experiments that will measure the effect on the activity of cholinergic interneurons of distinct inputs arriving at discrete dendritic sites.

The kyphotic curve is the primary convex shaped curvature of the spine and is an essential component of optimal thoracic function. In females aging and osteoporosis contribute to significant increases in thoracic kyphosis. However studies describing thoracic posture in young women are few, with findings largely based on measurements taken from radiographs. The present study investigated thoracic posture and global and segmental angles in healthy young women using non-invasive technology.

A skin-surface computerised accelerometer (SpinalMouse®) was used to register spinal curvature in the sagittal plane in upright standing, and end-range flexion and extension. Anthropometric measures for height and weight were taken from 21 female participants (age 26.5 ± 2.9y, mean ± SD). Following a warm-up exercise, two sets of spinal registrations were recorded in each of the three standardised postures by wheeling the accelerometer down the length of the spine from cervical 7 to lumbar 5 spinous processes. Descriptive statistics for anthropometric data and thoracic angles were analysed and used to describe potential positional differences of the spine for each of the kyphotic angles.

Global thoracic kyphosis was 41.5 ± 9.60, which is within the normal range (30-500). The greatest segmental kyphosis occurred at thoracic 7/8 in both upright standing and extension and at thoracic 9/10 in maximal flexion.

The results are clinically relevant as studies in older women have shown the thoracic apex to be at thoracic 5/6 in upright standing. There is thus a transitional change in kyphotic curvature with age which has not previously been recognised. Secondly, maximum kyphotic curvature is not fixed and changes with extremes of posture.

Registration of changes in spinal curvature using SpinalMouse® may assist in the recognition of increased thoracic kyphosis beyond the normal range. This approach could also be used to monitor the effect of interventions designed to minimise postural changes.

DNA gyrase, a topoisomerase required for bacterial DNA replication, is an important antibacterial target that is sensitive to the fluoroquinolone drugs. The emergence of drug resistance and of bioterrorism has generated fresh interest in discovering new drugs that can target this enzyme. The two subunits of the enzyme form a functional A2B2 complex but a crystal structure of the gyrase complex bound to its DNA substrate is lacking. This study aims to reconstitute purified subunits of DNA gyrase from the thermophilic archaebacterium Thermophilus thermus with a preferred DNA substrate, as a prelude to crystallographic analysis for structure-directed drug discovery.

Hexa-histidine tagged T. thermophilus DNA gyrase A and B subunits were separately cloned and expressed in Escherichia coli. The subunits were extensively purified by heat treatment of cell lysates at 65°C, nickel affinity chromatography and size exclusion chromatography. The DNA supercoiling activity of the reconstituted subunits was assayed by monitoring the conversion of relaxed plasmid pBR322 to its supercoiled form. The purified subunits were reconstituted with a suitable 130 bp DNA fragment of pBR322 (obtained by PCR) in the presence of Mg-ATP and the gyrase inhibitor novobiocin. The resultant complex was analysed using size exclusion chromatography and blue gel polyacrylamide electrophoresis.

The supercoiling activity of DNA gyrase was optimal at the expected subunit molar ratio of 1:1 and was inhibited by the 130 bp fragment of pBR322. Reconstitution of the protein subunits with the DNA fragment gave a novobiocin-stabilised complex larger than either subunit A or B. Formation of the moderately stable gyrase-DNA complex appeared to require novobiocin but not Mg-ATP.

Preliminary evidence has been obtained for the reconstitution of a novobiocin-stabilised DNA-gyrase complex. Future studies will identify conditions that more effectively stabilise the gyrase-DNA complex. Crystallographic analysis may then help identify gyrase inhibitors with novel modes of action.

Tutin is a natural convulsant compound found in the New Zealand native tutu tree, and others of the Coriaria genus worldwide. Unpublished experiments from our laboratory show significant suppression of γ-aminobutyric acid type A receptor (GABAA) currents by this compound, resulting in epileptiform activity in cultured neurons. Other research showing that 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reduces tutin-induced seizures has suggested that tutin is a convulsant through a direct activation of glutamate (particularly α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)) receptors. This project directly addresses this hypothesis.

Whole cell patch-clamping in primary cortical neuron cultures was employed. Excitatory currents were evoked, using either AMPA or n-methyl-D-aspartate (NMDA), to select for glutamate receptor superfamilies. Agonists were applied directly onto the cell soma for 500 msec at the concentration evoking 50% of the maximal response (EC50) (8 µM AMPA and 10 µM NMDA). In all cells we measured evoked currents before, during and after toxin perfusion (10 µM). For each cell we averaged the amplitude of the currents at steady-state. We then carried out two-tailed paired Student t-tests (P < 0.05 significant).

Tutin did not significantly alter AMPA or NMDA receptor currents. Control amplitudes for 10 µM NMDA (n = 5) were 1381 ± 373 pA (mean ± SEM). The addition of tutin resulted in average amplitudes of 1351 ± 860 pA (P = 0.5481). For 8 µM AMPA (n = 5), control readings averaged to 1662 ± 226 pA. Tutin resulted in 1781 ± 230 pA events, again not significantly different (P = 0.1085). We conclude that tutin does not exert its excitatory effects by modulating ionotropic glutamate receptors.

Plasma matrix metalloproteinase (MMP) and tissue inhibitors of matrix metalloproteinases (TIMP) levels have previously been implicated in the cardiovascular mortality of patients with coronary artery disease. This study aimed to determine if MMP-related markers were associated with a history of in-stent restenosis (ISR). The strength of any such associations was compared with that of other known demographic and clinical risk factors for ISR.

The circulating levels of three MMP-related markers (proMMP-9, active MMP-9 and TIMP-1) were assessed in 164 patients with in-stent restenosis (ISR) and compared with 168 patients with no history of ISR. Multiple logistic regression was used to determine the independence of associations. Multiple receiver operating characteristic (mROC) analysis was used to determine sensitivity and specificity of both individual and composite risk markers.

Univariant analysis indicated that both active MMP-9 and TIMP-1 were significantly elevated in ISR patients, whereas pro-MMP-9 was not. Active MMP-9 >2 ng per mL was associated with an adjusted odds ratio of 4.6 (95% confidence interval 2.4-9.1, P < 0.0001) for ISR. Analysis of conventional ISR risk factors by mROC resulted in a c-statistic of 0.839; 83.9% of cases were ranked higher than controls. Addition of MMP-related markers increased the c-statistic to 0.870, a 3.7% increase in the model’s discrimination. A cut down model, suitable for clinical risk prediction, yielded a c-statistic of 0.858 with MMP-related markers adding 5% to the model.

Plasma MMP-related protein levels are elevated in patients with a history of ISR. Addition of MMP-related proteins to currently accepted risk variables may increase the overall predictive value for ISR.