The utility of osteon shape and circularity for differentiating human and non-human Haversian bone

Distinguishing human from non‐human bone fragments is usually accomplished by observation of gross morphology. When macroscopic analysis is insufficient, histological approaches can be applied. Microscopic features, like plexiform bone or osteon banding, are characteristic of non‐humans. In the absence of such features, distinguishing Haversian bone as either human or non‐human proves problematic. This study proposes a histomorphometric approach for classifying species from Haversian bone. Two variables, osteon area (On.Ar.) and circularity (On.Cr.), are examined. Measurements were collected from three species (deer, dog, human) represented by various skeletal elements; only ribs were available for humans (ribs: deer n = 6, dog n = 6, human n = 26; humeri: deer n = 6, dog n = 6; femora: deer n = 6, dog n = 6). Qualitative analysis comparing human to non‐human On.Ar. demonstrated that human ribs have larger mean On.Ar. (0.036 mm²) than non‐human ribs (deer = 0.017 mm², dog = 0.013 mm²). On.Cr. in the ribs showed minor differences between species (deer = 0.877; dog = 0.885; human = 0.898). Results demonstrated no significant difference across long bone quadrants in long bones. Discriminant analyses run on the means for each sample demonstrated overlap in deer and dog samples, clustering the non‐human and human groups apart from each other. Mean On.Cr. proved a poor criterion (ribs only: 76.3%, pooled elements: 66.1%), while mean On.Ar. proved useful in identifying human from non‐human samples (ribs only: 92.1%, pooled elements: 93.5%). When variables were combined, accuracy increased to 100% correct classification for rib data and 98.4% when considering data from all elements. These results indicate that On.Ar. and On.Cr. are valuable histomorphometric tools for distinguishing human from non‐human Haversian bone. Am J Phys Anthropol, 2012. © 2012 Wiley Periodicals, Inc.     

Relationship between stomatal frequencies,yield components and morphological characters in collections of winter wheat cultivars

Genotypes on the Belgian national list as well as about 120 genotypes of winter wheat from the world collection were examined for the relationships of their stomatal frequencies in the flag leaf with yield, yield components and other morphological characters. Stomata were in rows, with a higher density in adaxial leaf surface. Cultivars differed significantly in both stomatal density and number of rows of stomata per unit of leaf width. Yield and morphological characters in general showed no significant correlation with stomatal frequencies. Densities in all series examined ranged from 46 to 78 stomata per mm² in the adaxial leaf epidermis and from 32 to 93 in the abaxial one.     

Mast cell heterogeneity in the human uvea

To identify chymase- and tryptase-positive mast cells in the human uvea, and to study their associations with different types of resident uveal cells, uveal specimens from 24 human donor eyes were cryosectioned in sagittal and tangential planes. Enzyme histochemical staining of chymase was combined with immunohistochemical staining for tryptase, detected with the APAAP method. Fluorescence immunohistochemistry was performed with antibodies against c-kit, alpha smooth muscle actin, protein gene product (PGP) 9.5, CD45, and HLA-DR. In different uveal compartments, the total amounts of mast cells were calculated and the distributions of chymase and tryptase were quantified. All uveal mast cells were c-kit and CD45 positive and HLA-DR negative. No association existed between mast cells and actin-containing cells. Only a few mast cells were in close association with PGP 9.5-labeled nerve fibers. In the choroid, most mast cells were located in the inner central part (mean density = 48.9/mm²), and contained both chymase and tryptase (96%). The ciliary muscle contained numerous mast cells (mean density = 33.7/mm²), many of them tryptase positive but chymase negative (63%). In the pars plana, a high number of chymase-positive, tryptase-negative mast cells were found (20%). In the iris only a few mast cells were present. Although the choroid contains the most common subtype of mast cells, a unique situation concerning the distribution of chymase and tryptase is present in the anterior uveal tissues. A possible role for these cells in the special immunological situation of the anterior eye chamber merits further investigation. Accepted: 16 September 1999     

Profile of native N‐linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time‐of‐flight mass spectrometry

Protein glycosylation involves the addition of monosaccharides in a stepwise process requiring no glycan template. Therefore, identifying the numerous glycoforms, including isomers, can help elucidate the biological function(s) of particular glycans. A method to assess the diversity of the N‐linked oligosaccharides released from human serum without derivatization has been developed using on‐line nanoLC and high resolution TOF MS. The N‐linked oligosaccharides were analyzed with MALDI FT‐ICR MS and microchip LC MS (HPLC–Chip/TOF MS). Two microfluidic chips were employed, the glycan chip (40 nL enrichment column, 43×0.075 mm² i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140×0.075 mm² i.d. analytical column), both with graphitized carbon as the stationary phase. Both chips offered good sensitivity and reproducibility in separating a heterogeneous mixture of neutral and anionic oligosaccharides between injections. Increasing the length and volume of the enrichment and the analytical columns improved resolution of the peaks. Complex type N‐linked oligosaccharides were the most abundant oligosaccharides in human serum accounting for ∼96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining ∼4%.     

Scaling of Haversian canal surface area to secondary osteon bone volume in ribs and limb bones

Studies of secondary osteons in ribs have provided a great deal of what is known about remodeling dynamics. Compared with limb bones, ribs are metabolically more active and sensitive to hormonal changes, and receive frequent low‐strain loading. Optimization for calcium exchange in rib osteons might be achieved without incurring a significant reduction in safety factor by disproportionally increasing central canal size with increased osteon size (positive allometry). By contrast, greater mechanical loads on limb bones might favor reducing deleterious consequences of intracortical porosity by decreasing osteon canal size with increased osteon size (negative allometry). Evidence of this metabolic/mechanical dichotomy between ribs and limb bones was sought by examining relationships between Haversian canal surface area (BS, osteon Haversian canal perimeter, HC.Pm) and bone volume (BV, osteonal wall area, B.Ar) in a broad size range of mature (quiescent) osteons from adult human limb bones and ribs (modern and medieval) and various adult and subadult non‐human limb bones and ribs. Reduced major axis (RMA) and least‐squares (LS) regressions of HC.Pm/B.Ar data show that rib and limb osteons cannot be distinguished by dimensional allometry of these parameters. Although four of the five rib groups showed positive allometry in terms of the RMA slopes, nearly 50% of the adult limb bone groups also showed positive allometry when negative allometry was expected. Consequently, our results fail to provide clear evidence that BS/BV scaling reflects a rib versus limb bone dichotomy whereby calcium exchange might be preferentially enhanced in rib osteons. Am J Phys Anthropol 151:230–244, 2013. © 2013 Wiley Periodicals, Inc.     

Heterogeneity of human natural killer cell populations.

Natural killing (NK) in human donors was determined by the ability of peripheral blood subpopulations to lyse the myeloid target, K562, in a 2 to 4 hr ⁵¹Cr release assay. The most active cell was a non-T cell which passed through nylon columns (representing 10 to 25% of column passed cells). A second column passed cell population, with characteristics of T lymphocytes (75 to 90% of column passed cells), was also capable of mediating natural killing. Non-T cells which were retained by the nylon columns (45 to 55% of adherent cells) lacked NK activity. However, nylon adherent T cells (45 to 55% of adherent cells) were consistently active in NK assays, illustrating an important subset of NK effector cell often overlooked. Both column passed and adherent T cells were further separated according to their ability to bind IgG or IgM immune complexes, showing that those mediating NK have receptors for IgG (Tγ+) but not for IgM (Tμ+).     

Residual stress distribution in rabbit limb bones

The presence of the residual stresses in bone tissue has been noted and the authors have reported that there are residual stresses in bone tissue. The aim of our study is to measure the residual stress distribution in the cortical bone of the extremities of vertebrates and to describe the relationships with the osteon population density. The study used the rabbit limb bones (femur, tibia/fibula, humerus, and radius/ulna) and measured the residual stresses in the bone axial direction at anterior and posterior positions on the cortical surface. The osteons at the sections at the measurement positions were observed by microscopy. As a result, the average stresses at the hindlimb bones and the forelimb bones were 210 and 149 MPa, respectively. In the femur, humerus, and radius/ulna, the residual stresses at the anterior position were larger than those at the posterior position, while in the tibia, the stress at the posterior position was larger than that at the anterior position. Further, in the femur and humerus, the osteon population densities in the anterior positions were larger than those in the posterior positions. In the tibia, the osteon population density in the posterior position was larger than that in the anterior position. Therefore, tensile residual stresses were observed at every measurement position in the rabbit limb bones and the value of residual stress correlated with the osteon population density (r=0.55, P<0.01).     

Estimation of age at death from second metacarpals.

This study examined the estimation of age at death from the second metacarpal in 227 individuals aged 30-98 years. Variables ascertained from each bone were: cortical thickness and microdensitometric cortical bone density measured on radiographs of the bone and total osteon count and density recorded on microradiographs of the complete cross section at its midshaft. Based on the latter two variables, two age groups were formed; a middle age group representing those individuals aged 30-65 years, and an older group aged 65+. Stepwise regression analysis of the four variables produced a series of regression equations for age estimation for the middle, old and combined age groups for each sex and sexes combined. Sex-specific equations provided better results than nonspecific ones, especially in females. Total osteon density and combined cortical thickness were found to be the most useful estimators in the middle and the old age group, respectively. The standard error of estimate was 6.71 and 6.90 years in each age group for the sexes combined. In the combined age group, age could be estimated accurately from total osteon count, cortical thickness and MD cortical bone density with the standard error of estimate of 11.10 years. The relative error of estimate ranged within +/- 30% in almost all individuals aged above 60 years.     

Determination of the numerical density of perforated synapses in rat neocortex

Summary The numerical density and frequency of perforated synapses in the molecular layer of rat parietal cortex have been determined using 4 procedures in an attempt to overcome problems associated with the size and complex three-dimensional shape of perforated synapses. The following procedures were compared: A, single-section analysis; B, adjacent-section analysis; C, semi-serial-section analysis; and D, complete serial-section analysis. All procedures made use of an unbiased counting rule.Estimates of the numerical density of perforated synapses ranged from 0.06 to 0.27×10⁹ mm^-3, and that of all synapses (non-perforated and perforated) from 1.88 to 2.50×10⁹ mm^-3. The frequency of perforated synapses varied from 4.5 to 18.0%. Procedures B (adjacent-section analysis) and D (complete serial-section analysis), neither of which utilize assumptions regarding the shape of synapses, produced comparable results (numerical density of perforated synapses 0.19–0.27×10⁹ mm^-3, and of all synapses 2.24–2.45×10⁹ mm^-3; frequency of perforated synapses 8.6–10.9%). The frequency of perforated synapses appeared to be underestimated by procedure A (single section analysis; 4.5%) and overestimated by C (semi-serial section analysis; 18%).It is concluded that adjacent-section analysis is the most efficient and effective procedure for determining the numerical density and frequency of complex particles, such as perforated synapses. There is, however, no significant difference in the performance of this procedure compared with that of single-section analysis, for determining the numerical density of synapses in general. Nevertheless, inherent problems of bias within the single-section procedure make the adjacent section method the procedure of choice.     

Proliferating cells in the rat anterior pituitary during the postnatal period: immunoelectron microscopic observations using monoclonal anti-bromodeoxyuridine antibody

Proliferating cells in the male rat anterior pituitary at 1, 3, 5, and 8 weeks of age were labeled with bromodeoxyuridine (BrdU) and studied by light and electron microscopic immunocytochemistry using anti-BrdU. They decreased in number from 402±31/mm² at 1 week to 50±1.5/mm² at 8 weeks, while their cell area increased by about twofold during this period. They had a slightly higher nucleus/whole cell (N/C) ratio than non-proliferating cells. According to their ultrastructure we classified them into granular and agranular cells. The percentage of granular cells ranged from 73% to 82% of all the proliferating cells during the period studied. They had many granules of various sizes and shapes, and some contained growth hormone and prolactin. Agranular cells, constituting 18–27% of proliferating cells, were small and had a high N/C ratio, indicating their immaturity. Moreover, they showed several features of folliculo-stellate (FS) cells: they showed no secretory granules in the cytoplasm, extended thin cytoplasmic processes, and sometimes they constructed a follicle among them. These results suggest: (1) the majority of proliferating cells were mature cells producing anterior pituitary hormone(s) and (2) most of the agranular proliferating cells maybe FS cells. The possibility of the latter is discussed.     

Retinal ganglion cell topography and spatial resolution in the smelt Hypomesus japonicus (Brevoort, 1856)

The present study deals with the topography of retinal ganglion cells (GCs) and spatial resolution in the smelt Hypomesus japonicus. The eyes and retinae were examined by light microscopy and computerized tomography. DAPI labelling was used to visualize cell nuclei in the ganglion cell and inner plexiform layers. Two zones of increased GC density in the nasal and temporal retina were bridged by a horizontal streak with the GC density ranging from 5600 to 8000 cells/mm². The maximum cell density (area retinae temporalis) ranged from 9492 to 14,112 cells/mm², and the total number of GCs varied from 286 x 10³ to 326 x 10³ cells in three individuals. The theoretical anatomical spatial resolution (the anatomical estimate of the upper limit of visual acuity) was minimum in the ventral periphery (smaller fish, 1.43 cpd; larger fish, 1.37 cpd) and maximum in area retinae temporalis (smaller fish, 2.83 cpd; larger fish, 2.41 cpd). The relatively high density of GCs and presence of the horizontal streak and area retinae temporalis in the H. japonicus are consistent with its highly visual behaviour. The present findings contribute to better understanding of the factors affecting the topography of retinal ganglion cells and mechanisms of visual adaptation in fish.     

Therapeutic Hypothermia Modifies Perinatal Asphyxia-Induced Changes of the Corpus Callosum and Outcome in Neonates

What Is Known about this Subject?

Diffusion-weighted MRI has demonstrated changes in the corpus callosum of term neonates with perinatal asphyxia. The severity of cerebral changes demonstrated using diffusion-weighted MRI is difficult to assess without measuring values of the Apparent Diffusion Coefficient (ADC).

What Is New?

ADC values of the anterior part of the corpus callosum are slightly higher than of the posterior part in full term infants with perinatal asphyxia. Low ADC values of the corpus callosum were associated with an adverse outcome in infants with perinatal asphyxia. In infants treated with hypothermia lower ADC values than with normothermia were associated with a poor outcome, supporting neuroprotective effects of hypothermia

Background

Using MRI, changes can be detected in the corpus callosum (CC) following perinatal asphyxia which are associated with later neurodevelopmental outcome.

Aim

To study the association between the apparent diffusion coefficient of water (ADC) in the CC on MRI in neonates with perinatal asphyxia and neurodevelopmental outcome at 18 months of age.

Subjects, Methods

Of 121 infants 32 (26%) died and 13 (11%) survived with an adverse neurological outcome. Sixty-five (54%) received therapeutic hypothermia. MRI was performed within 7 days after birth using a 1.5 T or 3.0 T system, and ADC values were measured in the anterior and posterior CC. The association between ADC and composite outcome (death or abnormal neurodevelopment) was analyzed for both normothermia and hypothermia cases using receiver operating characteristics.

Results

ADC values of the posterior CC were lower than of the anterior part (mean difference 0.050 x 10^-3 mm²/s, p<0.001). Field strength did not affect ADC values. ADC values of the posterior part of the CC were significantly lower in infants with basal ganglia/thalamus or near total brain injury (p<0.001). Lower ADC values were associated with an adverse outcome, but cut-off levels were lower after hypothermia (1.024 x 10^-3 mm²/s vs 0.969 x 10^-3 mm²/s)

Conclusion

Low ADC values of the posterior part of the corpus callosum are associated with an adverse outcome in term or near term neonates with perinatal asphyxia. Therapeutic hypothermia slightly modifies this association, showing that lower values were needed for an adverse outcome.     

Phytoplankton assemblage structure and dynamics as indicator [-2pt] of the recent trophic and biological evolution of the western basin of Lake Como (N. Italy)

We studied the phytoplankton assemblage of the western basin of Lake Como (Northern Italy) during 1997. The phytoplankton assemblage was composed of 65 taxa, belonging to six taxonomic groups. Chlorophyta were represented by the highest number of taxa (28) followed by Bacillariophyceae (17), Cyanoprokaryota (9), Dinophyceae (6), Chrysophyceae (3) and Cryptophyta (2). The total assemblage density and biomass ranged from 902 ind ml^–1 and 134.5 mm³ m^–3 in February to 58 766 ind ml^–1 and 9360 mm³ m^–3 in October. The density and biomass variation at three stations showed a common pattern, with higher values in the southern part of the basin where TP concentrations were always greater. The phytoplankton succession was analysed by cluster analysis (average linkage clustering) and non-metric multidimensional scaling (NMDS) ordination, both applied to a dissimilarity matrix obtained from a calculation of the Bray–Curtis index. In general, the seasonal succession followed a simple pattern, with a clear spring phase. These results are discussed considering the trophic evolution of the lake and its recent colonization by Dreissena polymorpha.     

Interfacial Strength of Cement Lines in Human Cortical Bone

In human cortical bone, cement lines (or reversal lines) separate osteons from the interstitial bone tissue, which consists of remnants of primary lamellar bone or fragments of remodeled osteons. There have been experimental evidences of the cement line involvement in the failure process of bone such as fatigue and damage. However, there are almost no experimental data on interfacial properties of cement lines in human cortical bone. The objective of this study is to design and assemble a precision and computer controlled osteon pushout microtesting system, and to experimentally determine the interfacial strength of cement lines in human cortical bone by performing osteon pushout tests. Thirty specimens were prepared from humeral diaphyses of four human subjects. Twenty specimens were tested under the condition of a small hole in the supporting plate, in which the cement line debonding occurred. The cement line interfacial strength ranged from 5.38 MPa to 10.85 MPa with an average of 7.31±1.73 MPa. On the other hand, ten specimens were tested under the condition of a large hole in the supporting plate, in which the shear failure inside osteons was observed. The specimens tested under the condition of the large hole resulted in an average shear strength of 73.71±15.06 MPa, ranging from 45.97 MPa to 93.74 MPa. Therefore, our results suggest that the cement line interface between osteon and interstitial bone tissue is weaker than that between bone tissue lamellae.     

New sonographic measures of peripheral nerves: a tool for the diagnosis of peripheral nerve involvement in leprosy

To evaluate ultrasonographic (US) cross-sectional areas (CSAs) of peripheral nerves, indexes of the differences between CSAs at the same point (∆CSAs) and between tunnel (T) and pre-tunnel (PT) ulnar CSAs (∆TPTs) in leprosy patients (LPs) and healthy volunteers (HVs). Seventy-seven LPs and 49 HVs underwent bilateral US at PT and T ulnar points, as well as along the median (M) and common fibular (CF) nerves, to calculate the CSAs, ∆CSAs and ∆TPTs. The CSA values in HVs were lower than those in LPs (p < 0.0001) at the PT (5.67/9.78 mm²⁾ and T (6.50/10.94 mm²⁾ points, as well as at the M (5.85/8.48 mm²⁾ and CF (8.17/14.14 mm²⁾ nerves. The optimum CSA- receiver operating characteristic (ROC) points and sensitivities/specificities were, respectively, 6.85 mm² and 68-85% for the PT point, 7.35 mm² and 71-78% for the T point, 6.75 mm² and 62-75% for the M nerve and 9.55 mm² and 81-72% for the CF nerve. The ∆CSAs of the LPs were greater than those of the HVs at the PT point (4.02/0.85; p = 0.007), T point (3.71/0.98; p = 0.0005) and CF nerve (2.93/1.14; p = 0.015), with no difference found for the M nerve (1.41/0.95; p = 0.17). The optimum ∆CSA-ROC points, sensitivities, specificities and p-values were, respectively, 1.35, 49%, 80% and 0.003 at the PT point, 1.55, 55-85% and 0.0006 at the T point, 0.70, 58-50% and 0.73 for the M nerve and 1.25, 54-67% and 0.022 for the CF nerve. The ∆TPT in the LPs was greater than that in the HVs (4.43/1.44; p <0.0001). The optimum ∆TPT-ROC point was 2.65 (90% sensitivity/41% specificity, p < 0.0001). The ROC analysis of CSAs showed the highest specificity and sensitivity at the PT point and CF nerve, respectively. The PT and T ∆CSAs had high specificities (> 80%) and ∆TPT had the highest specificity (> 90%). New sonographic peripheral nerve measurements (∆CSAs and ∆TPT) provide an important methodological improvement in the detection of leprosy neuropathy.     

Visualisation of capillaries in human skeletal muscle

 A double staining method combining Ulex europaeus agglutinin I lectin (UEA-I) and collagen type IV staining was used to determine the capillary density and the number of capillaries relative to different fibre types in human skeletal muscles. The result of this combined staining was compared with that of other staining methods including amylase-periodic acid Schiff (PAS), UEA-I, anti-collagen type IV and anti-von Willebrand factor. Muscle biopsy specimens, 12 from M. vastus lateralis and 6 from M. soleus, were obtained from 18 healthy young men. Compared with amylase-PAS staining, double staining showed a larger number of capillaries surrounding type I (+9.6%), type IIA (+8.6%) and type IIB (+11.6%) fibres in the M. vastus lateralis specimens (P<0.001 for all differences). The capillary to fibre ratio (cap·fibre^–1) and the capillary density (cap·mm^–2) were 8.3% (P<0.002) and 7.9% (P<0.001) larger, respectively. In the M. soleus specimens, cap·fibre^–1 and cap·mm^–2 were 7.4 and 9.9% larger, respectively, by double staining compared with PAS staining. Further comparisons showed that the cap·fibre^–1 and cap·mm^–2 obtained with double staining were similar to the values determined by the UEA-I staining, but greater than that measured by the collagen type IV method. The double staining gave a more marked stain of capillaries and revealed muscle fibre borders clearly, which is an advantage in studies that require comparisons between serial sections using computerised image analyses. It is concluded that the double staining method is superior to either the UEA-I, collagen type IV or the traditional amylase-PAS staining methods in analysing capillary density of normal human skeletal muscle. Accepted: 17 October 1996     

Microglial cells of the rat brain in postnatal period (comparative immunocytochemical analysis)

In the course of the brain’s development, distribution of microglial cells was studied in rats using immunocytochemical detection. To identify the microglial cells, antibodies to lipocortin 1 (LC1) and phosphotyrosine (PT) were used. On postnatal day 1, LC1-positive microglial cells of an ameboid shape were distributed mainly in the subventricular zone; their mean density was 31±8 cells/mm² (counted across the total area of frontal sections). On postnatal day 7, microglial cells of an intermediate type were located throughout the whole brain; their density was 54±15 cells/mm². On the 15th day, LC1-positive cells were of a ramified shape, and their density reached 104±20 cells/mm² (the microglial cell density in the mature normal brain was 103±3 cells/mm²). On postnatal day 7, PT-positive cells were similar in their morphology to LC1-positive cells of an intermediate type, while their mean density was 32 cells/mm². In the mature brain, the density of PT-positive microglia was 53±5 cells/mm²; the shape of the cells in the white and gray matter of the brain was, on the whole, similar to that of LC1-positive microglia. Therefore, LC1 is a specific marker for different types of microglial cells in the developing brain. Our data about 3D distribution and morphological peculiarities of microglial cells at different stages of postnatal development are consistent with the hypothesis on the neuroectodermal origin of microglia.     

Longitudinal zonation and life cycles of macrozoobenthos in the Mauerbach near Vienna,Austria

A total of 12277 benthic invertebrates were caught from November 1990 to October 1991 at the Mauerbach, a first to fourth order forest brook near Vienna, Austria, using a Surber sampler and a hand net. Five taxa comprised 96.6% of the catch: Diptera larvae (45.6%), Amphipoda (37.6%) and larvae of Ephemeroptera (6.4%), Plecoptera (5.3%) and Trichoptera (1.7%). Mean values of macrozoobenthos density ranged from 39514 specimens m⁻² at upstream sampling site 5 in December to only 286 specimens m⁻² at sampling site 3 in May, shortly after a severe flood. Based on benthic invertebrate population structure, sites 5 and 6 (situated near the source) were clearly separated from the downstream sites 1 to 4. At upstream sites, shredders comprised up to 71.7% of the total, whereas at downstream sites collectors were most abundant. In addition, the proportion of eucrenal species decreased from 12% at site 6 near the source to only 2% at site 1 near the mouth. Among the insect species studied in detail, most were univoltine except Ephemera danica (Ephemeroptera) and Sericostoma personatum (Trichoptera), which had a two-year life cycle.     

Functional domains of bovine β-1,4 galactosyltransferase

A number of N- and C-terminal deletion and point mutants of bovine -1,4 galactosyltransferase (-1,4GT) were expressed inE. coli to determine the binding regions of the enzyme that interact withN-acetylglucosamine (NAG) and UDP-galactose. The N-terminal truncated forms of the enzyme between residues 1–129, do not show any significant difference in the apparentK _ms toward NAG or linear oligosaccharide acceptors e.g. for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose. Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose. From these columns the protein can be eluted with 15mm NAG and 50mm EDTA, like the enzymatically active protein, TL-GT129, that contains residues 130–402 of bovine -1,4GT. Also the N-terminus fragment, TL-GT129NAG, that contains residues 130–257 of the -1,4GT, binds to, and elutes with 15mm NAG and 50mm EDTA from the NAG-agarose column as efficiently as the enzymatically active TL-GT129. Unlike TL-GT129, the TL-GT129NAG binds to UDP-columns less efficiently and can be eluted from the column with only 15mm NAG. The C-terminus fragment GT-257UDP, containing residues 258–402 of -1,4GT, binds tightly to both NAG- and UDP-agarose columns. A small fraction, 5–10% of the bound protein, can be eluted from the UDP-agarose column with 50mm EDTA alone. The results show that the binding behaviour of N- and C-terminal fragments of -1,4GT towards the NAG- and UDP-agarose columns differ, the former binds preferentially to NAG-columns, while the latter binds to UDP-agarose columns via Mn²⁺.     

Comparison of N(2) Fixation and Yields in Cajanus cajan between Hydrogenase-Positive and Hydrogenase-Negative Rhizobia by In Situ Acetylene Reduction Assays and Direct N Partitioning

Pigeon peas [Cajanus cajan (L.) Millsp.] were grown in soil columns containing ¹⁵N-enriched organic matter. Seasonal N₂ fixation activity was determined by periodically assaying plants for reduction of C₂H₂. N₂ fixation rose sharply from the first assay period at 51 days after planting to a peak of activity between floral initiation and fruit set. N₂ fixation (acetylene reduction) activity dropped concomitantly with pod maturation but recovered after pod harvests. Analysis of ¹⁵N content of plant shoots revealed that approximately 91 to 94% of plant N was derived from N₂ fixation. The effect of inoculation with hydrogenase-positive and hydrogenase-negative rhizobia was examined. Pigeon peas inoculated with strain P132 (hydrogenase-positive) yielded significantly more total shoot N than other inoculated or uninoculated treatments. However, two other hydrogenase-positive strains did not yield significantly more total shoot N than a hydrogenase-negative strain. The extent of nodulation by inoculum strains compared to indigenous rhizobia was determined by typing nodules according to intrinsic antibiotic resistance of the inoculum strains. The inoculum strains were detected in almost all typed nodules of inoculated plants.

Gas samples were taken from soil columns several times during the growth cycle of the plants. H₂ was never detected, even in columns containing pigeon peas inoculated with hydrogenase-negative rhizobia. This was attributed to H₂ consumption by soil bacteria. Estimation of N₂ fixation by acetylene reduction activity was closest to the direct ¹⁵N method when ethylene concentrations in the gas headspace (between the column lid and soil surface) were extrapolated to include the soil pore space as opposed solely to measurement in the headspace. There was an 8-fold difference between the two acetylene reduction assay methods of estimation. Based on a planting density of 15,000 plants per hectare, the direct ¹⁵N fixation rates ranged from 67 (noninoculated) to 134 kilograms per hectare, while grain yields ranged from 540 to 825 kilograms per hectare. Grain yields were not increased with N fertilizer.